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So I've been working as a donation attendant at a certain thrift store, which strangely enough, I freaking love. It's a really nifty job because I get to sort through people's stuff that they may or may not have wanted to donate.
And we get some really weird stuff. For realz.
But there was one day that was particularly interesting to say the least.
I was in the donation area of the outside of the building, catching some sun. Enjoying the absolutely horrifically hot weather. When a guy drove up, his wife in the car, and he practically shoves this HUGE box into my arms.
"Thank you for your donation sir," I said to him, muffled from behind the box.
"We just got out of the military and we don't need our kitchenware anymore, so I figure you guys can use it," he said, giving me a wolfish grin and walking back to his car, where his lovely wife was playing on her smartphone.
I thought nothing of this. Very normal. The box was a big plastic bin with the lid and it was marked "KITCHENWARE" so I figured that there was nothing fishy about it.
So I brought it to where we sort the donations and handed it off to a volunteer that was working that day. He took it, anxious for something to do, and I began to walk off when I heard him call out to me.
"Uh, Ms. Leah? I think you need to see this."
I walk over and I realize that this man did not donate a box full of kitchenware.
He donated a box full of sex toys.
And I don't mean a little box with two or three things. I mean, he donated a huge tote of sex toys.
And it had everything you could and could not imagine
The box that was donated to us had costumes, whips, dildos, vibrators, different sexy games, lingerie… basically anything and everything you could imagine.
Me and the volunteer just stared at the box for a few minutes, unsure of what to do next.
Finally, I decide it would be a good idea to tell my manager.
While he was in the room with two very prude women.
Who were also managers.
As you can imagine, the results were hysterical. Too funny for words. To say the least, the entire situation was horribly awkward.
So to date, that is the best donation story I've got from working at a thrift store for six weeks.


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Knowing how to fix a leaky faucet is one of many skills mastered by the professionals at Action Auger.
The majority of all faucet leaks are caused by washers that have seen better days. To replace that worn, improperly installed or ill-fitting washer that is causing your faucet to leak may require a quick trip to the hardware store to pick up a few essentials… if, in fact, that is the problem.
Call your local Action Auger plumber for help or more information on how to fix a leaky faucet.
Dedicated to serving the needs of our residential clients, Action Auger is home to some of the best plumbing technicians in the industry.
Address: 3638 Manchester Road SE, Calgary, AB Canada
Phone: (403) 879 1663
Email: see contact us
Members enjoy amazing discounts and font-of-the-line service – Learn more.

subset: commoncrawl


text: Hip-hop and skateboarding, that's what summer fun is made of. This Saturday, Stash magazine and FTC team up to bring you a free show at Potrero Del Sol Skatepark. The lineup should curl your toes: San Quinn, Pep Love, Opio, Balance, Rick Flare, Bored Stiff, Mistah F.A.B., Beetiki, K.O.S., Spank Pops, Bash Bros., and J-Billion, with DJs Haylow and Ant One on the decks. That's what's up, all you rail grinders!


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Questions? Contact the event organizer listed below.
- This event has passed.
May Flowers
May 4, 2019 @ 5:00 am - 10:00 am
Event Navigation
Spring is in the air and our vendors are ready to show you all their Spring inventory. Come shop with 30+ local crafters and vendors to get your homes ready for Spring and Summer. Do not forget to grab all those gifts for Dad's, Grad's, Baby Showers, Wedding Showers, and more. Admission is FREE.
If you have any questions or are interested in being a vendors please email us at firstname.lastname@example.com


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Consulting is the act of giving expert advice to people working in a professional or technical field to a third party for a fee.
Consulting is most often used when a company needs an outside, expert opinion regarding a business decision. Example, a company wishing to sell its products in another country may hire a consultant with knowledge of the business practices of that country.
The consultant will inform the company about the best practices that should be followed, what to expect from customers, and how to deal with foreign regulations.
Consulting can come in many flavours like technical, legal, international, security (electrical or physical), management (education, accountancy, law, human resources, marketing and public relations.
Consultants can give their advice in a variety of forms. Often reports and presentations are used, but in some specialised fields, the consultant may use (or build) customised software or product for the client. In some cases the advice can be made public by putting it online, but in many cases it remain the sole property of the client.
Where can you find consultants in the UK ? below will be a list of some of the more well known ones :-
- Consultancy UK is an advisory and consulting industry platform for UK-based consultants. The website has the latest news and trends in the consulting sector, follows the developments and publications of consulting firms across 60+ industries and functional areas and provides an overview of career opportunities for professionals interested in working in consultancy.
- List of consulting firms in London, UK is a good resource if you need a consultant based in or around the city.
- HR Bridge Consulting Ltd is an Anglo-French consultancy based in London that provides solutions to international companies who put Human Resources in the heart of their strategy and decisions. They specialise in post-Brexit consultation (advice on how to turn Brexit to your advantage), international HR operations, employment law in practise, corporate restructuring, HR strategy and project management.
- Infinity Group IT Consultancy has six offices in the UK: Kent, Sussex, Surrey, Birmingham and two in London, one is located in The Shard, central London.
- Clutch has compiled a list of the top 426 IT consultants based in London.
- FT Research has compiled a list of the UK's Leading Management Consultants 2019: ratings and analysis.
(C) 2016 ~ 2020 SI7 - Stats


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Skill Life Fun Cooking Breakfast Fast-food
Cook the food as people order as quick as possible. If food is not done and being drag out, it has to be thrown to the rubbish bin and cooked again. $50 will be deducted for wastage. Once food is dragged on the plate/bowl, you can not move them no more.
Use mouse to interact.


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Are you ready for the 2017 recession?
"'Nominal' GDP growth is now 2.4% – the lowest rate, outside of recession, since World War II..."
Source: Daily Crux
http://thecrux.com/are-you-ready-for-th ... recession/
The Institute for Supply Management's ("ISM") index of manufacturing unexpectedly dipped again last month. It fell to 49.4 in August, down from 52.6 in July. (Readings below 50 indicate contraction, while readings above 50 indicate growth.)
According to official statistics, more than 9 million net new jobs have been created in the U.S. since 2005. But according to Katz and Krueger, all of these jobs have been in "alternative work."
These are temporary, on-demand, and/or independent-contracting jobs that pay less, provide no benefits, and offer fewer and less-reliable hours compared with traditional work.
Does the participation rate today look anything like the dot-com boom that actually raised almost everyone's boat at least a bit? Short answer: No., it doesn't.
Wages as a percentage of GDP has been in a 45-year freefall that can only be described as Depression for wage earners
Is the economy in a Depression? Not if you're a corporate bigwig skimming vast gains from corporate buybacks funded by the Fed's free money for financiers.
But if you're a wage earner who's seen your pay, hours and benefits cut while your healthcare costs have skyrocketed–well, if it isn't a Depression, it's a very close relative of a Depression.
Users browsing this forum: Google [Bot] and 1 guest


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Our Ultra-Sil™ Duffle Bag is the ultimate in lightweight bags. With an incredible capacity of 40 Litres and strong enough for a heavy load, it is the perfect bag as an airline carry-on, for impromptu excursions, or just going to the gym. Super compact and super light, it is an obvious choice as a spare bag on any trip.
Great little containers for organizing all your condiments, liquid toiletries and more! HDPE constructed HDPE is a rigid, high tensile strength material that is UV...View full product details
By using up-cycled foam off-cuts from Self Inflating Sleeping Mats, our new Foam Core Pillow reduces waste. The soft and stretchy 50D knit face fabric...View full product details
Sea To Summit Lightweight 70D Dry Sack A great allrounder - less expensive and less bulky alternative to traditional dry bags. You can squash these...View full product details


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A 49-year-old resident of Walvis Bay tragically died when she was trapped in a fire that gutted 18 shacks.
The incident occurred on Sunday evening in Sandwich Street, Kuisebmond in Walvis Bay.
The woman, who was identified as Laimi Iyambo, reportedly left a candle burning in the shack that she shared with her daughter. However the daughter was not home when the inferno erupted.
Shocked onlookers and residents said the fire started very quickly and spread to nearby shacks, resulting in the 18 shacks being destroyed by the fire.
When New Era arrived on the scene huge flames engulfed sections of the shacks. Some of the owners of the shacks scrambled around to save their belongings that could be seen scattered on the pavement. Paramedics and the fire brigade arrived shortly afterwards and tried at least for an hour to contain the fire, after which the charred remains of Iyambo were removed from the scene.
Several victims were treated for shock and smoke inhalation on the spot while some were taken away to receive further medical treatment.
According to the fire chief of the Walvis Bay Municipality, Willie Basson, it is believed the deceased and some friends were drinking earlier that day and that she apparently went back to her shack to rest after her friends had left. However, several minutes later smoke was seen coming from her shack. The neighbours tried to warn the deceased but the fire became too intense.
In a different incident the Namibian Police in Swakopmund are looking for a suspect accused of murdering his roommate in Mondesa on Saturday evening close to the Vrede Rede School.
According to Inspector Erastus Iikuyu the suspect allegedly stabbed his 27-year-old roommate Steven Eichab to death after returning from a drinking spree.
Iikuyu said the two, who were accompanied by a friend shared a room and apparently argued over food. This resulted in the suspect stabbing Eichab who died minutes later.
Source : New Era


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ShowingFilter 1-3 of 3 listings.
Snorkeling and Island Tours
$ 60
3 Hours Snorkeling Free Cancellation
Explore the islands of Balboa Province during a 2-hour excursion from Isla Contadora. With your local guide, travel by boat to the sandbar at Boyarena, from which you can see a dozen surrounding tropical islands. At Chapera Island, jump in to go snorkeling on the reef among a plethora of tropical fish and other marine life. Continue to Mogo Mogo Island, a remote, rugged location where the television show 'Survivor' was filmed. Relax in the shallow lagoon before returning to your departure point. Use of snorkel equipment and snacks are provided.View Tour
Belize Hol Chan Marine Reserve and Shark Ray Alley Snorkel Tour from Ambergris Caye
$ 67
3 Hours Snorkeling Hotel Pickup Offered Free Cancellation
Explore the underwater wonders of Belize's most popular snorkeling sites with this 3-hour trip to Hol Chan Marine Reserve and Shark Ray Alley. Departing from Ambergris Caye, you'll swim with an amazing variety of fish, sea turtles, stingrays, and nurse sharks among the coral formations of the largest barrier reef in the Western Hemisphere.View Tour
Snorkeling Tour from Roatan Island, Honduras
$ 36
2 Hours Snorkeling Hotel Pickup Offered Free Cancellation
See the marine life around Roatan Island in Honduras even if you don't SCUBA dive. This half day tour from West Bay Beach includes a boat ride to a site offshore for snorkeling over part of the largest barrier reef in the Caribbean and the chance to see reef fish and more. Continue to Anthony's Key for more snorkeling and the chance to see dolphins.View Tour


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Interested in joining the Minnesota Oldsmobile Club? Click here for more details.
Oldsmobile Club of America
For more information on the OCA or to find a chapter near you, visit the OCA Web site.
Axel's River Grill
1318 Sibley Memorial Highway
Mendota Heights, MN
6:00pm - 7:00pm Social Hour
7:00pm - 9:00pm Dinner
9:00pm - 10:00pm Annual Membership Meeting


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Motorists stop traffic allowing gaggle of geese to cross Canadian highway
A group of motorists on an Edmonton, Alberta highway halted traffic to allow a gaggle of Canadian Geese to cross multiple lanes of traffic.
Captured on Sunday (July 7) at Whitemud and Henday from Sherwood Park, drivers already on the highway as well as those joining traffic slow down to let the birds advance across the road.
The filmer said: "As all Canadians do, we stop and let them safely pass."
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Volt Workforce Solutions is NOW HIRING 2nd Shift Packers!
No hablas ingles, le podemos ayudar.
Location: Mountville, PA
Hours: 3:00 - 11:00 pm
Pay rate: $10.75/hour
Call Yazmin Rodriguez for more information 717-742-1128
If you want to get your foot on the door with a company in the food manufacturing industry and your ultimate goal is to secure permanent employment, we have the job for you!
No experience required! We offer a temp to hire PT or FT job opportunity based on attendance and performance.
Job Requirements:
**Hablamos Espanol**
Volt has over 60 years of staffing experience. We work with many of the Fortune 500 and 1000 companies to provide workforce solutions. We offer our workers competitive pay and benefits.
Come work in an employee friendly environment with a diverse workforce.
Volt is an EEO employer.


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Free Shipping on your entire order! Code: FREESHIP
Restrictions Apply | See All Offers
Toys, books & more inspired by exploration and discovery
The first edition of Ultimate Weird but True was an instant hit, and now the second edition has even more zany fun! Ultimate Weird but True 2 has a fun cover with lots of sparkle and is packed with cool photos!
The Science of Everything takes readers on a fascinating tour, using plain talk, colorful photography, instructive diagrams, and everyday examples to explain the science behind all the things we take for granted in our modern world.
A colorful picture book featuring inspirational messages from beloved Disney and Disney-Pixar films, The Magic Is in You focuses on the perseverance and resilience of cherished characters and encourages readers of all ages to believe in themselves in any situation.
This latest addition to the crazy popular Weird But True series is an exciting internal journey through your own wacky, weird, sometimes gross, but always fascinating human body.
How does a rainbow form? What causes it to snow? What makes the wind blow? This adorable reference book introduces young children to every aspect of weather and answers the questions curious kids want to know!
Chock-full of puzzles, optical illusions, cranial challenges, and information on the latest research in neuroscience, this awesome activity book helps you discover even more about your amazing brain! It's kid-friendly fun, based on the National Geographic hit television show Brain Games.
Packed with science, puzzles, and tons of fun, this Brain Games: Big Book of Boredom Busters activity book based on the hit National Geographic television show will fire up your neural network!


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High Risk Offender wanted on warrants
The Calgary Police Service is seeking public assistance to locate a high risk offender who is wanted on warrants.
Kevin Ronald BROOKS, 28, was released into the Calgary area on Tuesday, June 23, 2015, after serving a seven-year federal prison sentence for manslaughter.
BROOKS is currently wanted on three warrants for failing to comply with conditions of his release by not residing at an approved location, failing to abide by a daily curfew check and not wearing a court approved electronic monitoring device.
In the early hours of Friday, Sept. 4, 2015, officers attended his approved residence when an alarm sounded to indicate his monitoring device had been removed. They were unable to locate BROOKS who has not been seen since.
BROOKS also has a history of violent offences. He has been violent against strangers, people known to him and peace officers. His past offences have been committed in Edmonton, Drumheller and Saint John, NB.
BROOKS is described as Caucasian, approximately 160 pounds, 6' tall, with brown hair and brown eyes. He also has multiple tattoos, including one around his neck, and tear drops by the corner of his right eye.
A photo of BROOKS is available on The City of Calgary Newsroom.
Members of the public are advised that the intent of the process is to enable members of the public to take suitable precautionary measures and not to embark on any form of vigilante action.
Anyone who has information about the whereabouts of BROOKS is asked to call police at 403-266-1234 or Crime Stoppers anonymously using any of the following methods:
TYPE: www.calgarycrimestoppers.org
TEXT: tttTIPS to 274637
15373177/15373205 / 4157


text: Size: L Color: Dark Gray PAWZ Road are committed to offer cozy life for your furry baby. KEY FEATURES: 1.Our cozy blanket is lightweight that is easy for your furry baby to carry around 2.Made for premium plush fibers that provided warmth for snuggling and snapping. 3.Easily to shake off pets fur and machine washable . 4.Multiple color and colorful dots look great and matches all kinds of home decors. 5.Available in FOUR size: Small , Medium, Large and Extra Large Specification Color:Grey Blue,Pink and Dark Gray Material: Micro Fibers Size Info.: S:20X30, M :40x30, Large:55 X40 XL:78X59 Weight:S:100g, M:208g, L:400g XL:750g


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Department of Public Works
From Highway Department Chief Robert Smith
The past several months we, gratefully, have seen an exceptionally mild winter. To date, we have plowed
only 7 times, but completed 28 rounds of street salting. The milder winter has allowed us to complete
some indoor projects, including the repainting of Village Hall and the Fire Hall bathrooms. The mild winter
has also provided the DPW the opportunity to clean and flush over 3000 linear feet of sanitary sewer lines
and we have undertaken the task of switching out our traditional fluorescent light fixtures in our Highway
Garage to efficient and cost-saving LEDs.
Within the next few weeks, construction work will begin on our new salt storage facility, located at the foot
of Roland Street near the cell tower site. Our rock salt has been stored in the rear garage of the
Community Center but over the years, has caused some damage due to its corrosive nature. Delivery at
the Community Center is difficult too, as the overhead door limits the type of transfer truck that can be
utilized and interferes with school employee parking.
As spring is around the corner, we would like to remind everyone that while mowing their lawns, to not
blow the clippings into the street. During rain, the clippings wash into the storm drain and create
blockages and potential backups. As residents complete their spring yard cleanup, remember that all yard
debris must be bagged and placed INSIDE of your trash tote except for branches, which can be either put
in your tote or bundled and left at curbside for retrieval by our department. If you need to dispose of any
old tires, for a few dollars each they can be recycled at Geiter-Done, located at 300 Greene Street in
Snowplow Contractors
must obtain a permit
before they can service any Village residence. Permits
cost $25.00 and are available from
Village Hall, 425 Reiman Street, 716-897-1560
, Mon-Fri between
the hours of 9:00 AM-4:00PM.
Residents are responsible for maintaining the sidewalk and headland (the strip between the curb and
the sidewalk) on their property
In the Fall, DPW will pick up leaves for residents. Bagging is
required. Simply pile the leaves on
the headland (the strip between the curb and the sidewalk) and DPW will vacuum them up.
Do NOT
pile leaves in the street!
To authorize DPW to
a tree
on the right of way, complete an
Authorization Form
mail to/drop it by Village Hall, 425 Reiman Street during regular business hours.
Forms may be
viewed/printed here


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Fanuc M-710i
Six-axis, modular construction, electric servo-driven robot designed for a variety of manufacturing and system processes. The innovative design of the FANUC M-710i provides an extremely large work envelope. Besides maximum reach, the double-jointed arm enables the M-710i to have the largest horizontal stroke in its class. Engineered for high speed and reliability, the M-710i delivers repeatable precision and flexibility with minimal maintenance.
The Fanuc R-J2 controller incorporates the most advanced and proven technology to improve total system performance while simplifying overall cell and system integration. The Fanuc RJ2 controller provides the optimal level of performance with any level of system requirements.
- AC servo motors with high power-to-weight ratios provide an extremely fast composite speed.
- Compact body castings provide the smallest Joint 2 interference zone in the industry.
- Wide axis ranges offer the largest horizontal strokes in this robot class.
- Fail-safe brakes on all six axes increase safety, functionality and control.
- Extremely rigid design allows precise positioning.
- Cables are routed through hollow RV reducers to minimize flexing.
Robot Info:
- Maximum Load of Robot: 45 Kg.
- Number of axis: 6
- Maximum Reach: 1706 mm.
- Repeatability: 0,15 mm.
- Controller:RJ2
- Structure:Vertical jointed-arm type
Robot Motion Speed:
- Axis 1: 160º/seg
- Axis 2: 120º/seg
- Axis 3: 150º/seg
- Axis 4: 240º/seg
- Axis 5: 240º/seg
- Axis 6: 340º/seg
Axis motion ranges
- Axis 1: 320/360º
- Axis 2: 260º
- Axis 3: 420º
- Axis 4: 540º
- Axis 5: 250º
- Axis 6: 720º
Arc welding , Palletising , Loading and unloading of parts , Handling of parts , Polishing , Assembly of parts


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Three schools in the Shannonside region are among 36 nationally that have been selected for a Hot School Meals pilot project to start in September.
Mohill National School in Leitrim, St Joseph's Convent National School in Longford town, St Anne's Convent National School in Castlerea, and Eoin Naofa National School in Ballymore in Co Westmeath are all among the schools chosen.
The project began as a pilot at a school in Inchicore at the beginning of this year and research from that showed that children who benefit from a daily nutritious hot dinner have greater focus and better learning outcomes.
Each school taking part in the project will be expected to provide a menu choice of at least two different meals a day per day plus a vegetarian or vegan choice, and an option that caters for student's religious and cultural dietary requirements.
Orla Egan, principal of St Josephs in Longfords, says it's wonderful boost for the school.


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Apostleship of the Sea of the United States of America (AOS-USA)
International Christian Maritime Association (ICMA)
International Seafarers Welfare and Assistance Network (ISWAN)
Mission to Seafarers
North American Maritime Ministry Association
Port Ministries International
Seamen's Church Institute of New York & New Jersey with links to the Center for Seafarers' Rights.
Maritime News
Fairplay Shipping news
Marine Log Shipping news
Newslink Online newspapers
Maritime News
International Labour Organization
Ship Talk
Maritime – General Info & Advocacy
Equasis Ship Search Ship data base: Name, Flag, Call Sign, Year Built, Type of Ship, etc.
Houston Maritime Museum
International Labour Organization
International Maritime Organization
International Transport Workers' Federation
Journal of Commerce
Philippine Overseas Employment Administration
Seafarers International Union
Canadian Coast Guard
Customs and Immigration Canada
International Transport Workers' Federation
MARAD (U.S. Department of Transportation Maritime)
Philippine Overseas Employment Administration
Port of Houston Authority
U.S. Coast Guard
U.S. Coast Guard (Houston-Galveston)
U.S. Customs and Border Protection
U.S. Department of State Background Notes: Factual publications that contain information on all the countries of the world with which the United States has relations. They include facts on the country's land, people, history, government, political conditions, economy, and its relations with other countries and the United States.
World Fact Book
American Diabetes Association
American Heart Association
Mamma Deep Web Health Search Engine
MedLine PlusService of U.S. National Library of Health and NIH
Country Calling Codes
Currency Converter
International Dialing Codes
Online Translator
Online Conversion
Seth Abramson's Criminal Justice
Time Zone Data Base


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Former NY City Transit Authority Contractor Employee Ordered to Pay $310,000 for False Statements
On May 4, Srinivas Thimmappa, a former project manager for Trataros Construction, Inc. (TCI), Brooklyn, NY, was sentenced in U.S. District Court in Brooklyn to pay $310,000 in fines and restitution for making false statements to federal agents. The New York Metropolitan Transportation Authority (NY-MTA), of which the NY-CTA is a part, is the FTA's largest grantee. TCI had been awarded a one-year job order construction contract with the NY-MTA with a $40 million ceiling, and Thimmappa's duties included subcontractor selection, approving subcontractor invoices, and submitting TCI invoices to NY-CTA. In November 2004, Thimmappa pled guilty to a charge of false statements made to investigators from DOT OIG, the General Services Administration's OIG and the FBI. Thimmappa claimed that in addition to his TCI employment, he performed part-time engineering work for a TCI subcontractor, Logix Construction Services (Logix), but denied having any other role in the company. Investigation found that Logix was a front company, established and used by Thimmappa under a false invoicing scheme to steal TCI funds. The ongoing investigation is being conducted jointly with GSA-OIG and the FBI.


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Politics and You
Ref: C2525478
Due to face to face teaching being suspended unfortunately this course is not currently available for enrolment.
We are launching a range of online courses shortly that may be of interest to you so sign up here to hear more.
Politics plays a fundamentally important role in our daily lives. With the imminent general election in mind, this course will explore how the issues around the election can affect us in the future in the way we live our lives.
Course aim
The course will explore how politics and political decisions affect our daily lives.
Do I need any particular skills or experience?
- No skills or experience needed
- This course is suitable for beginners and improvers
By the end of the course I should be able to:
- Have a better understanding of the role that politics play in our daily lives
- Use and interpret primary source material
- have confidence in participating in group discussion .
- Anding
How will I be taught?
- The WEA tutor will use a range of different teaching and learning methods and encourage you and the group to be actively involved in your learning
What kind of feedback can I expect?
- A range of informal activities will be used by the tutor to see what you are learning which may include quizzes, question and answer, small projects and discussion
- You will have opportunities to discuss your progress with your tutor
What else do I need to know?
- Nothing else is needed
- All materials will be provided
- A selection of materials and basic equipment will be provided but you are welcome to bring additional materials with you
Pre-course work, reading and information sources
- No pre reading is required but research on the subject on the internet or in the library may be helpful
What can I do next?
- Progress to another WEA course
- Progress to a course with another provider
- Become involved with the WEA in a range of voluntary work and other activities including campaigning as a WEA member
- Become involved as a volunteer for a WEA partner or another organisation
Download full course information sheet
Due to face to face teaching being suspended unfortunately this course is not currently available for enrolment.
We are launching a range of online courses shortly that may be of interest to you so sign up here to hear more.


text:
Honoring Indigenous Voices
Date & Time:
Wednesday, November 13, 2019 - 1 p.m. to 2:30 p.m.
Hagerty Lounge, 1928 St. Marys Road, Moraga, CA 94575
View a map and get directions.
View a map and get directions.
Join us in our screening and discussion of Waabooz and Fem 101, both written by Wenonah Wilms. Wilms won the title of 2018 Nicholl Fellow, for her award-winning screenplay Horsehead Girls. We invite all students to attend and hear her stories as an indigenous woman and writer. 1 p.m. Screening and Q&A in Hagerty Lounge.
At 6:30pm on Wednesday, Wenonah will be in the Center for Women & Gender Equity for the Coffee, Creativity and Community event where she will co-facilitate a writing workshop. Snacks and inspiration will be provided.
Add to Calendar 2019-11-13 13:00:00 2019-11-13 14:30:00 6 Honoring Indigenous Voices [[{"type":"media","view_mode":"media_original","fid":"810067","attributes":{"alt":"Honoring Indigenous Voices","class":"media-image no-caption","height":"445","id":"1","style":"float: left; width: 500px; height: 445px; margin-right: 10px;;;;;;;;"," https://prod.stmarys-ca.edu/honoring-indigenous-voices Hagerty Lounge, 1928 St. Marys Road, Moraga, CA 94575 DD/MM/YYYY
Sharon Sobotta


text:
Patrick Warburton Net Worth
How rich is Patrick Warburton? For this question we spent 7 hours on research (Wikipedia, Youtube, we read books in libraries, etc) to review the post.
The main source of income: Actors
Total Net Worth at the moment 2020 year – is about $30 Million.
Patrick Warburton information Birth date: November 14, 1964 Birth place: Paterson, New Jersey, United States Height:6 ft 2 in (1.905 m) Profession:Actor, Voice Actor, Model, Film Producer Nationality:United States of America Spouse:Cathy Jennings (m. 1991) Children:Alexandra Catherine Warburton, Gabriel Warburton, Shane Warburton, Talon Patrick Warburton Siblings:Lara Warburton, Mary Warburton, Megan Warburton
Height, Weight
:How tall is Patrick Warburton – 1,74m.
How much weight is Patrick Warburton – 53kg
Patrick John Warburton is an American actor. He is known for his several television roles, including the title role on The Tick, David Puddy on Seinfeld, Johnny Johnson on NewsRadio, Jeb Denton on Less than Perfect, and Jeff Bingham on Rules of Engagement. His distinctive de…
The information is not available
Wikipedia Source: Patrick Warburton


text:
There was plenty of reason for Leah Maunsell to celebrate when she ended the 2019 Enduro World Series as Under 21 Runner-Up.
The Irish National Enduro champion was a class act this year, leading a large part of the series.
Heading into the finale round in Zermatt, Switzerland, trailing points leader and fellow Kona rider, Lucy Schick, she faced a tall ask to overhaul her in the standings.
Although still learning the way of the Alpine stages, the Ballyhoura Trail Rider gave it her all for an eventual fourth – finishing four seconds behind Schick – to secure the runner-up result.
"It's been such an amazing year getting to race bikes in so many beautiful places around the world and I'm so grateful for that opportunity," told Leah. "It's taken so much help from many hands to get here, definitely too many to mention here.
"I'd just like to say a special thanks to Morrision BMW and Will Fogarty Construction for helping me get to this round and of course to my parents who are by far my greatest sponsors and supporters.
"Big up to whole Team Callaghan Clan for all their help this weekend and throughout the season too."
In Pro Men, Cube's Greg Callaghan signed off on his 2019 season with 18th overall. In what proved a season of injuries and misfortune, Greg ended his championship campaign 29th.
Claiming his best result of 2019, Specialized's Killian Callaghan ended Zermatt in 32nd to complete his first year in Pro Men in 45th.
Although still feeling the effects of his recent illness, Kelan Grant battled on for 55th to take 36th overall in 2019.
Bad luck hit Dan Wolfe in Switzerland ringing his bell in a heavy crash. At risk of concussion, he wisely opted to sit race day out.
70th overall in 2019, his year has most certainly got to be highlighted by riding to 50th in Whistler on a borrowed downhill bike. Epic!
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This morning I woke up to a pretty shitty message from someone (swipe to read). It's in reference to yesterday's @world_enduro and the fact that I called it after S2. I wouldn't usually put this sort of stuff up but it's shit to have a bad race and then read this. Yesterday I got pitched into a dried up river bed full of big ass rocks. I landed onto my head and shoulder. It was a painful impact and I didn't get up for a while. Once I got going my front brake was unusable and my derailleur a bit screwed. I finished the stage and decided to call it. To give you some perspective on this decision, earlier this year I got a concussion in the Les Orres EWS and continued to race on it until the riders and medics talked me into quitting. I hadn't really experienced a concussion before but instantly respected the severity of any head injury. With that knowledge I made a call yesterday, a call that isn't easy to make but needed to be made. Luckily I don't feel too shit today but I couldn't of known that yesterday. I listened to my gut. So you can imagine my annoyance when I read this message. I shouldn't need to prove anything to anyone but myself. I guess this guy didn't see what happened to my bike in Whistler this year (my bike fell off the ski lift 2 hours before my race so I raced the entire @world_enduro on a DH bike and came 50th). I'm where I am in the bike industry because I've put a crazy amount of years, energy, crashes, upsets, wins etc into this sport. You don't become a professional in this sport easily if you come from Ireland 😂 I'm coming to the end of my rant. To all you racers wanting to succeed in some shape or form in the bike industry, it's the years of dedication and determination that makes it pay off. Not just race wins (although they help 😂). Don't judge too quickly. There can be a lot more at play than what you see through Instagram. Not the way I planned the last @world_enduro to go but I'm happy I'm still in piece and made it through the 2019 season relatively injury free. I'm looking forward to lots of video projects and a healthy off season of training. @svenmartinphoto this pic is phenomenal 🍻
The Under 21 Men saw Gravity Enduro champ Harry Byrne come out swinging for 13th. Setting the sixth and seventh fastest times on stages one and two, Byrne had to be happy with that effort.
Behind him Drew Armstrong came in 25th, despite crashes on stages one and four.
Emmett Callaghan just missed out on a top-30 result with 31st.
With the 2019 Enduro World Series – won by Sam Hill and Isabel Courdurier – put to bed, attention now turns to the Trophy of Nations this weekend in Finale Ligure, Italy.
C'mon Ireland – 'Hup the parish!


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Usage of EM – Compost Rubbish
Insert the EM-Compost Rubbish inside the soil to activate the soil so as to allow healthy vegetables and flowers to be produced. Vitamins and minerals available in the EM-Compost Rubbish is enough to fertilize the crop and is free from usage of chemical fertilizer and inseticides.
- Vegetable crop in wide area
- Flowering plants crop in vases
Usage of EM – Rubbish Juice
- Pour in the toilet bowl, sink and drain to avoid clogging, rust and eliminate foul smell.
- Pour in the drain and it naturally cleans the sewage plant and river.
- Melted with water 500~1000X melting to be sprayed to the crop / flowering plant as a liquified fertilizer.
Any Enquiries can be forwarded to the address below:
Pusat Latihan Pengembangan Mesra Alam
Mengendalikan Kursus Asas Pengenalan Dan
Aplikasi Bioteknologi EM Untuk
Pejabat Pendaftaran :
No. 17-01, Jalan Sagu 15, Taman Daya, 81100 Johor Bahru, Johor. Malaysia.
Tel : 607 – 353 4997
Fax : 607 – 355 4997
Email : firstname.lastname@example.com
No. 10 Jalan Kemajuan, Batu 24, Taman Industri Kota Tinggi, 81900 Kota Tinggi, Johor, Malaysia.
Tel/Fax : 607 – 882 3997


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Behavioral based interviewing as a method to identify the best qualified applicant for the job
March 2, 2020
Mis-hires can cost an organization dearly in ways that can be both qualitatively and quantitatively measured.
Behavioral based interviewing can help ensure that the best qualified applicant is identified during the recruiting process.
Please watch the following brief video on how to develop behavioral-based interviewing questions.
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About The Laureate
This mid 19th centaury mansion, steeped in history and National Cape Monument status rests in Gardens, behind the historic Mount Nelson Hotel.
Fine dining is the order of the day, at the only venue in the world where you can enjoy cuisine excellence in the company of four Nobel Peace Prize Laureates commissioned oil portraits, hung on the historical walls.
Discover a rare dining experience, steeped in history and breathtaking surrounds at The Laureate at Leinster Hall.
+27 (0) 21 424 1836
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The Rad4 ATR-Activation Domain Functions in G1/S Phase in a Chromatin-Dependent Manner
DNA damage checkpoint activation can be subdivided in two steps:
initial activation and signal amplification. The events distinguishing these two phases and their genetic determinants remain obscure. TopBP1, a mediator protein containing multiple BRCT domains, binds to and activates the ATR/ATRIP complex through its ATR-Activation Domain (AAD). We show that Schizosaccharomyces pombe Rad4TopBP1 AAD–defective strains are DNA damage sensitive during G1/S-phase, but not during G2. Using lacO-LacI tethering, we developed a DNA damage–independent assay for checkpoint activation that is Rad4TopBP1 AAD–dependent. In this assay, checkpoint activation requires histone H2A phosphorylation, the interaction between TopBP1 and the 9-1-1 complex, and is mediated by the phospho-binding activity of Crb253BP1. Consistent with a model where Rad4TopBP1 AAD–dependent checkpoint activation is ssDNA/RPA–independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4TopBP1 AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4TopBP1 AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD–dependent Rad3ATR checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on protein–chromatin interactions.
Published in the journal:
. PLoS Genet 8(6): e32767. doi:10.1371/journal.pgen.1002801
Category: Research Article
doi: 10.1371/journal.pgen.1002801
DNA damage checkpoint activation can be subdivided in two steps:
initial activation and signal amplification. The events distinguishing these two phases and their genetic determinants remain obscure. TopBP1, a mediator protein containing multiple BRCT domains, binds to and activates the ATR/ATRIP complex through its ATR-Activation Domain (AAD). We show that Schizosaccharomyces pombe Rad4TopBP1 AAD–defective strains are DNA damage sensitive during G1/S-phase, but not during G2. Using lacO-LacI tethering, we developed a DNA damage–independent assay for checkpoint activation that is Rad4TopBP1 AAD–dependent. In this assay, checkpoint activation requires histone H2A phosphorylation, the interaction between TopBP1 and the 9-1-1 complex, and is mediated by the phospho-binding activity of Crb253BP1. Consistent with a model where Rad4TopBP1 AAD–dependent checkpoint activation is ssDNA/RPA–independent and functions to amplify otherwise weak checkpoint signals, we demonstrate that the Rad4TopBP1 AAD is important for Chk1 phosphorylation when resection is limited in G2 by ablation of the resecting nuclease, Exo1. We also show that the Rad4TopBP1 AAD acts additively with a Rad9 AAD in G1/S phase but not G2. We propose that AAD–dependent Rad3ATR checkpoint amplification is particularly important when DNA resection is limiting. In S. pombe, this manifests in G1/S phase and relies on protein–chromatin interactions.
The DNA damage checkpoint is an elaborate signal transduction pathway that monitors the integrity of the DNA, prevents cell cycle progression and promotes appropriate DNA metabolism [1] reviewed in [2]. The DNA damage sensors associated with checkpoint activation define two separate DNA structure-dependent signal transduction cascades. Each pathway engages a phospho-inositol-3 kinase-like protein kinase (PIKK); either the Ataxia Telangiectasia Mutated (ATM) or the Ataxia Telangiectasia and Rad3 related (ATR) kinase [3]. ATM detects DNA double strand breaks (DSBs) by interaction with the Mre11-Rad50-Nbs1 repair complex, while ATR primarily senses single stranded-DNA (ss-DNA) through interactions with RPA. Both ATM and ATR are conserved in the model organisms S. pombe and S. cerevisiae.
For ATR to recognise a DNA lesion, single-stranded DNA (ssDNA) needs to be formed - for example by DNA repair-dependent DNA processing [4] or following the replication machinery encountering the unrepaired lesion [5]. Once ssDNA is generated, it is immediately coated by replication protein A (RPA) (Reviewed in: [6]). Multiple ATR molecules are initially recruited to ssDNA regions via ATRs obligate binding partner, ATRIP, which binds directly to RPA [7], [8]. ATR-ATRIP recruitment to ssDNA-RPA is necessary for "basal" ATR activation, but is insufficient for full checkpoint activation: co-recruitment of a second complex consisting of three PCNA-like proteins, Rad9, Hus1 and Rad1 (known as the 9-1-1 clamp) is also necessary. 9-1-1 is loaded in parallel to ATR recruitment at 5′ ssDNA/dsDNA junctions by the checkpoint clamp loader Rad17-RFC[2–5] [9], [10], [11]. (Figure 1A).
When ATR-ATRIP is first loaded at the site of ssDNA, its "basal" kinase activity promotes phosphorylation of its immediate neighbours, including ATRIP [12], [13], an in trans phosphorylation of a residue within ATR itself, T1989 [14], and the subunits of the 9-1-1 clamp [15], [16]. Dependent on the concomitant recruitment of 9-1-1, a further protein, TopBP1, is recruited [17]. TopBP1 is recruited via an interaction between its BRCT (1+2) domains and a constitutive phosphosphorylation on the C-terminus of Rad9 [18], [19]. Similarly in both yeast systems, Saccharomyces cerevisiae and Schizosaccharomyces pombe, the TopBP1 homologs, Dpb11 and Rad4 respectively, are recruited by the phosphorylation of the C-terminus of Rad9Ddc1 creating a binding site for a pair of BRCT domains (Figure 1A). In S. pombe, the C-terminal phosphorylations occur on Rad9 at residues T412 and S423 [20]. This subsequently recruits Rad4TopBP1 via interaction with BRCT pair (3+4). However, unlike in mammalian cells, T412 and S423 in S. pombe are directly targeted by Rad3ATR in response to its ssDNA/RPA binding and concomitant 9-1-1 loading [16], [20], [21]. Despite these differences, in both S. pombe [20] and mammalian cells [14], Rad4TopBP1 recruitment promotes the formation of a Rad3ATR/9-1-1/Rad4TopBP1 complex (Figure 1A). However, the mode of interaction of Rad3ATR and Rad4TopBP1 within this complex has not been defined in S. pombe.
Mammalian TopBP1 can directly activate ATR-ATRIP both in vitro in the absence of ssDNA/RPA and when over-expressed in cells. TopBP1-dependent ATR activation requires an ATR activation domain (AAD) situated between the 6th and 7th BRCT domains [17] and mutation of a conserved aromatic residue within this unstructured region, W1147, prevents this mode of ATR activation. The AAD contacts a region within the C-terminus of ATR, between the kinase and FATC domains [22], which has been termed the PIKK Regulatory Domain (PRD). Mutation of a conserved PRD residue, K2598, similarly abolishes TopBP1-dependent ATR activation. In both mammalian cells and Xenopus extracts the interaction between TopBP1 and ATR-ATRIP appears to be essential for checkpoint activation in response to replication stress [17], [22], although the initial in trans phosphorylation of ATR on T1989, reportedly essential for full ATR activation, is TopBP1-independent [14].
In the budding yeast model system, the intrinsically disordered C-terminal extension of the TopBP1 homolog, Dpb11TopBP1, contains an AAD which interacts with and activates Mec1ATR via a pair of aromatic residues, W700 and Y735 [22], [23], [24], [25]. Interestingly, in S. cerevisiae, at least two distinct Mec1ATR activation domains have been identified: in addition to the Dbp11TopBP1 AAD, the C-terminal tail of Ddc1Rad9 (S. cerevisiae homolog of the 9-1-1 subunit Rad9) contains an AAD that can directly stimulate Mec1ATR activity in vitro and contributes to checkpoint activation in vivo [11], [26]. The key residues in Ddc1Rad9 required for Mec1ATR activation are W352 and W544. W352 resides on the surface of the PCNA-like domain, while W544 lies within the intrinsically disordered C-terminus. In vivo the Ddc1Rad9 AAD is essential for Mec1ATR activation when S. cerevisiae cells are in G1 [24], [26], while the Ddc1Rad9 AAD acts redundantly with the C-terminal AAD of Dpb11TopBP1 during checkpoint activation in G2. It is proposed that, at least in S. cerevisiae, a minimum of one other protein contains an equivalent AAD (Reviewed in [27]).
We have previously shown that S. pombe Rad4TopBP1 is not required for the Rad3ATR-dependent and DNA damage-dependent phosphorylation of Rad26ATRIP or the 9-1-1 clamp subunits [16], [20], demonstrating that Rad3ATR is active at sites of DNA damage in the absence of activation by the Rad4TopBP1 AAD. However, the presence of Rad4TopBP1 is clearly required to form a robust Rad3ATR/9-1-1/Rad4TopBP1 complex [20], to recruit the Crb253BP1 mediator protein [28], [29] and for Rad3ATR to phosphorylate downstream substrates such as Chk1-S345 [30] and promote robust checkpoint activation.
To further explore the role of Rad4TopBP1 in checkpoint activation in S. pombe we identified and characterised the Rad4TopBP1 AAD. We show that Rad4TopBP1 can interact with Rad3ATR via its AAD and that the AAD contributes to Rad3ATR activation in vivo. We observe that the biological function of the Rad4TopBP1 AAD is most important in G1/S phase, when resection is limited, and that reducing DSB resection in G2 following ionising radiation results in compromised Chk1 phosphorylation in the absence of Rad4TopBP1 AAD function. In order to separate out and study Rad4TopBP1 AAD-dependent Rad3ATR activation we developed a Rad4TopBP1 AAD-dependent lacO-LacI checkpoint activation system for S. pombe and used this to show that Rad4TopBP1 AAD-dependent Rad3ATR activation is also dependent on histone H2A phosphorylation. Consistent with a role for this chromatin modification, mutations in Crb2 that interfere with phospho-binding by Crb2 also decrease Rad4TopBP1 AAD-dependent checkpoint activation. Thus, the Rad4TopBP1 AAD-dependent Rad3ATR activation pathway is chromatin dependent, implying a role in checkpoint amplification and maintenance.
In S. cerevisiae, two aromatic residues, W700 and Y735 were identified as critical for Dbp11TopBP1 AAD activity [24], [25]. We similarly created an alignment of Rad4TopBP1 with the C-terminal tails of a variety of TopBP1 homologs and identified a short sequence encompassing Y599 in S. pombe (Figure 1B, 1C), which we subsequently confirmed as defining the AAD activity of Rad4TopBP1 (see below). To characterise the function of the Rad4TopBP1 AAD, we separately mutated the conserved aromatic amino acid to create strain rad4-Y599R or deleted the minimal conserved motif to create rad4-Δ[595–601].
To establish if the Rad4TopBP1 AAD interacts with Rad3ATR we expressed and purified recombinant Rad4[288–648] (that includes BRCT (3+4) and the unstructured C-terminal tail) as a fusion with GST. Constructs containing a mutation in either the AAD (Y599R), deletion of residues 643–645 encompassing a putative cyclin binding motif (RxL), or mutation of a CDK phosphorylation site (S641A) were similarly purified. Wild-type and all the mutated recombinant proteins, when incubated with yeast extracts, bound to and co-purified the control interacting partner of Rad4TopPB1, Cdc13CyclinB (unpublished data). Wild-type Rad4-GST also co-purified Rad3ATR-Myc from extracts prepared from 3myc-rad3 cells (Figure 1D). In contrast, the amount of Rad3ATR-Myc pulled-down with recombinant AAD-mutated Rad4TopBP1-GST was reproducibly reduced (n = 4). Neither Rad3ATR-myc nor Cdc13CyclinB were pulled-down with a GST-only control. We conclude that, in vitro, this residue of Rad4TopBP1 is part of a Rad4TopBP1 interaction domain for Rad3ATR, consistent with it having the properties of an AAD. We also note that the interaction with Cdc13CyclinB is not abolished by loss of the RxL motif and that mutation of S641 affects the Rad4TopBP1:Rad3ATR interaction in vitro (Figure 1D, lane 4).
rad4-AAD mutants show checkpoint defects associated with S phase
Both rad4-Y599R and rad4-Δ[595–601] displayed normal cell cycle progression (data not shown), indicating that, as is observed in S. cerevisiae [24], the Rad4TopBP1 AAD is not required for unperturbed DNA replication. In response to UV, MMS and HU treatment, rad4-Y599R and rad4-Δ[595–601] cells showed intermediate sensitivity when compared to rad4+ and checkpoint defective rad3Δ (Figure 1E and Figure S1A). To establish if the sensitivity to DNA damage correlated with a defective G2 DNA damage checkpoint, we monitored cell cycle progression after cells were synchronised in G2 and UV-irradiated. Following exposure to 50 Jm-2 (Figure 2A), rad4-Y599R cells displayed premature release from cell cycle arrest (∼20 min earlier than rad4+ after 50 Jm-2). We next monitored the sensitivity and checkpoint response to ionising radiation. rad4-Y599R mutant cells displayed only very mild sensitivity to IR (Figure 2B) and the checkpoint was mildly extended (Figure 2C). We do not know the reason for the slight extension of the G2 delay: no obvious increase in numbers or duration of Rad22Rad52 foci were observed, indicating no significant delay to DSB repair (Figure S1B).
We next examined Chk1 phosphorylation status in rad4+ and rad4-Y599R cells as a surrogate for checkpoint activation (Figure 2D). In response to 200 Jm-2 UV irradiation, asynchronously growing rad4-Y599R cells displayed reduced Chk1 phosphorylation when compared to rad4+ cells, consistent with the partial checkpoint defect observed. Conversely, in response to IR, no significant difference is seen between rad4-Y599R and rad4+. An upstream target of Rad3ATR is the C-terminus of histone H2A [31], [32]. To establish if the UV-specific defect in Rad3ATR-dependent phosphorylation is specific to Chk1, we monitored γH2A formation following either UV or IR treatment (Figure 2E). As was seen for Chk1 phosphorylation, a significant decrease in γH2A is observed in rad4-Y599R cells following UV but not IR treatment when compared to rad4+ cells.
The pattern of DNA damage sensitivity seen for rad4-Y599R cells is consistent with a specific sensitivity within S phase. >70% of fission yeast cells in an asynchronous culture are in G2 and mitosis is followed rapidly by S phase: G1 is extremely short. In response to IR, the G2 DNA damage checkpoint is robustly activated and DNA repair completed before cells pass through mitosis and into S phase [33]. Thus, following IR, relatively few cells replicate damaged DNA. Conversely, the G2 checkpoint is not robustly activated following UV [34] and the majority of UV-irradiated cells pass through mitosis and enter S phase with damaged DNA. To monitor S phase-specific events, we thus examined γH2A induction in cells treated with either hydroxyurea (HU), an inhibitor of ribonucleotide reductase, or Camptothecin (CPT), an inhibitor of topisomerase I (Figure 2F). Consistent with both agents manifesting cytotoxicity in S phase, γH2A levels were significantly reduced when comparing rad4-Y599R with rad4+ cells. Finally, since replication of UV damaged DNA induces Cds1Chk2 activity [35], we monitored the kinase activity of immuno-precipitated Cds1Chk2 following UV irradiation of rad4-Y599R and rad4+ cells (Figure 2G). Cds1Chk2 activation was reproducibly lower for rad4-Y599R, indicating an impaired S phase checkpoint activation (n = 3).
The rad4-AAD mutants are sensitive to DNA damage in S-phase
If the rad4-Y599R mutant is deficient in activation of Rad3ATR in S phase, we would anticipate increased sensitivity to IR within S phase when compared to rad4+ cells. To test this possibility, rad4-Y599R mutant and rad4+ cells where either synchronised in G2 cells using cdc25-22 or in G1 using a cdc10-m17. Following the block, cells were released by reducing the temperature and cell cycle progression was monitored by FACS analysis (Figure 3A, 3B). Cells were irradiated with 50 Gy IR at the times indicated. rad4-Y599R cells showed significant increased sensitivity when compared to rad4+ when irradiated in S phase, but not when irradiated in G2, when S phase is complete (i.e. see Figure 3B). In an equivalent cdc25-22 block and release experiment, we monitored Chk1 phosphorylation and γH2A induction (Figure 3C). Unlike when asynchronous rad4-Y599R cells are irradiated (>70% of such cells are in G2), when rad4-Y599R cells were irradiated in early-mid S phase, Chk1 phosphorylation was moderately reduced for the first 40 minutes after irradiation and γH2A levels were similarly decreased when compared to rad4+. Interestingly, following progression through S phase and into G2 (150 minute time point), Chk1 phosphorylation levels increased significantly in rad4-Y599R cells, although the same was not seen for γH2A levels.
To determine that the use of cdc25-22 synchronisation was not generating an artefact (Cdc25 is an activator of Cdc2-Cyclin B, which itself is required for normal DNA damage responses in G2 [36]), we used the alternative method of synchronisation where cells were arrested in G1 using cdc10-M17 and released directly into S phase (Figure 3D). Unlike IR treatment of asynchronous cultures where equivalent levels of γH2A were observed (Async IR), treatment of rad4-Y599R cells at 30, 60 or 90 minutes after release from arrest resulted in decreased γH2A levels and Chk1 phosphorylation when compared to rad4+ control cells.
LacO array-dependent checkpoint activation in S. pombe requires the Rad4TopBP1 AAD
In S. cerevisiae, co-localisation of two or more checkpoint proteins to arrays of lacO repeats bypasses the requirement for DNA damage in Mec1-mediated checkpoint activation [37]. To establish the role of the Rad4TopBP1 AAD in a what has previously been characterised as an RPA-ssDNA independent system, rad4TopBP1, rad9 and rad3ATR were each fused to a construct encoding GFP, the E. coli lac-repressor (LacI) and a nuclear localization signal (NLS); GFP/LN (Figure 4A). The resulting plasmids express the fusion construct under the control of a thiamine-repressible (nmt41) promoter. We established that each of the fusion constructs were functional by expressing them individually in the corresponding null mutants. Each was able to suppress the DNA damage sensitivity (and for rad4TopBP1, the thermosensitivity) of the appropriate mutant, although for rad4-GFP/LN genotoxin resistance was not restored to wild-type levels (Figure S1C–S1E). When expressed in cells harbouring 256 repeats of the lac operator sequence (lacO) integrated at the ura4+ locus, each fusion protein formed a single nuclear focus. No foci were detected in cells devoid of lacO arrays (Figure 4B).
We used Chk1 phosphorylation as a readout for DNA damage checkpoint activation (Figure 4C). Following thiamine removal (induction takes between 12 and 16 hours [38]), Chk1 became phosphorylated in lacO containing cells, but not in lacO-negative control cells, when either Rad3ATR, Rad4TopBP1 or Rad9 LacI fusion proteins were expressed. Similar results were obtained when each pair-wise combinations of two fusion proteins were expressed (Figure 5B). In S. pombe, DNA damage checkpoint activation results in cell cycle arrest and cell elongation. Elongated cells were observed upon expression of single fusion proteins (data not shown), confirming checkpoint activation. From these data we conclude that, in S. pombe, as in mammals [39] tethering of any of these single checkpoint proteins to a lacO array is sufficient to activate the DNA damage checkpoint and that, in contrast to the analogous experiments reported for S. cerevisiae, forced co-localisation of two checkpoint proteins is not required [37].
To establish if the Rad4TopBP1 AAD is involved in this damage-independent mode of checkpoint activation, we tested if Rad3ATR tethering could result in Chk1 phosphorylation in a rad4-Y599R mutant background (Figure 5A). While Rad3-GFP/LN expression resulted in induced Chk1 phosphorylation in rad4+ cells, Rad3-GFP/LN expression did not increase Chk1 phosphorylation in rad4-Y599R cells, demonstrating a role for the Rad4TopBP1 AAD. Next we established if expression and tethering of the AAD-defective Rad4-Y599R protein to lacO arrays was able to activate the checkpoint (Figure 5B). No induction of Chk1 phosphorylation was observed. Furthermore, while co-expression and tethering Rad3ATR and Rad9, or of Rad3ATR and Rad4TopBP1 resulted in checkpoint activation (Figure 5B), we observed that co-expression of Rad3ATR with Rad4TopBP1-Y599R mutant protein did not result in Chk1 phosphorylation. This data suggests that the AAD-defective mutant protein can act as a dominant negative, at least in this specific situation, preventing the endogenous wild-type Rad4TopBP1 from functioning with the tethered Rad3ATR to activate the checkpoint. It also supports the idea that the Rad4TopBP1 AAD domain is required for the activation of Rad3ATR and not simply recruiting it.
H2A phosphorylation is required for Rad4TopBP1 AAD–dependent checkpoint activation
While Rad3ATR kinase activity is essential for Chk1 phosphorylation in response to DNA damage [40], it also depends on the 9-1-1 clamp, the Rad17 clamp loader and the Crb253BP1 mediator. To characterise the dependencies for lacO-dependent checkpoint activation we examined which checkpoint genes were required for Chk1 phosphorylation during Rad3ATR tethering (Figure 5C). Rad3ATR-GFP/LN was expressed in lacO-positive strains deleted for rad1, rad9 (encoding 9-1-1 components), rad17 (clamp loader) and crb2. Each was required for Chk1 phosphorylation. Thus, Rad3ATR tethering is not sufficient for checkpoint activation: the clamp loader, the 9-1-1 clamp complex and the Crb2 mediator are all required and this artificial checkpoint activation system does not bypass the usual requirements. However, Brc1, the proposed MDC1/PTIP ortholog is not required for Chk1 phosphorylation in this system (Figure S1F)
In both S. pombe and S. cerevisiae recruitment of the 53BP1 ortholog (Crb2 and Rad9 respectively) to chromatin in response to IR requires prior phosphorylation of histone H2A [32], [41], [42]. In addition to H2A phosphorylation, recruitment also requires the largely constitutive methylation of a further histone residue, H3K79 in S. cerevisiae or H4K20 in S. pombe. These modifications are effected by distinct methylransferases in the two yeasts: Dot1 methylates H3K79 in S. cerevisiae [43] while Set9 methylates H3K20 in S. pombe [44], [45]. In S. pombe it has been demonstrated that the C-terminal BRCT domains of Crb253BP1 binds directly to γH2A [42] while the Tudor domain binds directly to di-methylated H3K20 [46]. Both interactions are required for Crb253BP1 chromatin association and show an epistatic relationship [45].
Using our Chk1 phosphorylation assay in response to Rad3ATR tethering, we tested two strains harbouring charge reversal mutations of residues within the phospho-acceptor site of the C-terminal Crb253BP1 BRCT domains, crb2-K617E and crb2-K619E (Figure 5C) that disrupt the interaction with γH2A [42]. Chk1 phosphorylation was reduced in both mutants. We next established if checkpoint activation by Rad3ATR tethering was affected in cells containing mutants in the two H2A genes that replace the phosphorylated residue with alanine, hta1-S129A hta2-S128A [32]. Chk1 phosphorylation was not observed in this background (Figure 5D), indicating that the Rad3ATR tethering-dependent and Rad4TopBP1 AAD-dependent checkpoint activation acts in the context of chromatin modification.
Rad4TopBP1 tethering bypasses the requirement for Rad9 C-terminal phosphorylation
Upon activation of the DNA damage checkpoint in S. pombe, Rad3ATR phosphorylates the Rad9 C-terminus on T412 and this is required to recruit Rad4TopBP1 [20]. Recruitment of Rad4TopBP1 allows subsequent recruitment of Crb253BP1 and consequent Chk1 activation [29]. A similar requirement for TopBP1 recruitment via Rad9 C-terminal phosphorylation is also evident in S. cerevisiae and mammalian cells [19], [47], [48]. As expected, expression of Rad3ATR-LacI in cells harbouring a rad9-T412A mutation did not result in Chk1 phosphorylation (Figure 5E) implying that lacO-recruited Rad3ATR must phosphorylate endogenous Rad9 to promote Rad4TopBP1 recruitment to activate the checkpoint.
We reasoned that the requirement for Rad9-T412 phosphorylation during activation by Rad3ATR tethering may solely be to bring the Rad4TopBP1 AAD into proximity of Rad3ATR. In this case, we should be able to bypass the requirement for Rad9-T412 phosphorylation specifically for checkpoint activation by Rad3ATR tethering by recruiting both Rad3ATR and Rad4TopBP1 at the same time. Indeed, Chk1 phosphorylation was restored when we co-expressed Rad3ATR-LacI and Rad4TopBP1-LacI in a rad9-T412A mutant background (Figure 5E). Since checkpoint activation by co-expression of Rad3ATR-LacI and Rad4TopBP1-LacI remains lacO dependent (Figure S1G), these data suggest that Rad4TopBP1 AAD can activate the Rad3ATR-dependent checkpoint cascade in the absence of the recruitment activity of the Rad9 C-terminal tail.
The Rad4TopBP1 AAD is important when resection is limiting
We have shown that the Rad4TopBP1 AAD functions to protect cells from insult during S phase, but is not required for G2 checkpoint activation after IR. Further, we demonstrated that when rad4-Y599R (AAD-defective) mutant cells were synchronised in S phase, phosphorylation of both Chk1 and H2A in response to IR treatment was reduced when compared to rad4+ cells. The increase in Cdc2-Cdc13ClyclinB (CDK) activity as cells progress from S phase into G2 [49] is known to establish conditions conducive to HR by regulating factors required for DNA resection [36], [50], [51]. A consequence of this is that, in response to IR treatment but not in response to UV treatment, ssDNA RPA is predicted to be more prevalent in G2 cells when compared to G1/S phase cells. We thus predicted that reducing resection rates associated with IR treatment in G2 cells would create a dependency for full Chk1 phosphorylation on the Rad4TopBP1 AAD and thus that Chk1 phosphorylation would be reduced in rad4-Y599R strains compared to rad4+ strains in response to an equal dose of IR.
To test this prediction we examined the induction of Chk1 phosphorylation in response to 100 Gy IR in exo1Δ rad4+ and exo1Δ rad4-Y599R cells (Figure 6). First we established that, when exo1 was deleted, Rad11RPA foci were reduced in number, consistent with the expectationt hat resection is decreased in this background (Figure 6A). In contrast to rad4+ cells, where exo1 deletion did not reduce Chk1 phosphorylation levels, Chk1 phosphorylation was reduced to approximately 50% when exo1 was deleted in AAD-defective cells. Previous work in both budding and fission yeasts has indicated that, in the absence of resection, Chk1 phosphorylation can occur through an alternative double strand break end-dependent Tel1ATM pathway, as opposed to the canonical resection and ssDNA/RPA-dependent Rad3ATR pathway [52]. Such a response could potentially mask some aspect of defects seen in the exo1Δ background. Thus, we first confirmed that loss of tel1 alone does not influence Chk1 phosphorylation in our assay (Figure S1H) and then concomitantly deleted Tel1ATM in both rad4+ and rad4-Y599R strains (Figure 6B, 6C). In the tel1Δ exo1Δ background, Chk1 phosphorylation was decreased by approximately 50% for both rad4+ and rad4-Y599R when compared to the exo1Δ alone background. These data are consistent with a general increase in Tel1-dependent checkpoint signalling when resection is reduced by exo1 deletion. In the background of the rad4-Y599R mutation, this is superimposed on a decrease in Rad3ATR-dependent signalling caused by the reduced resection.
The Rad4TopBP1 and Rad9 AADs co-operate in G1/S
A second ATR activation domain has recently been identified in the S. cerevisiae Ddc1Rad9 C-terminal tail [26]. Mutations in this domain define a function in Mec1ATR activation during G1, complementary to the function of the Dpb11TopBP1 AAD in promoting robust checkpoint activation in G2 in this organism [24]. Sequence alignments show that the two key Ddc1Rad9 AAD aromatic residues are conserved in S. pombe as Y271 (equating to W352Sc within the PCNA-like domain) and W348 (equating to W544Sc in the intrinsically disordered C-terminal tail) ([26] and Figure 7A, 7B).We thus created an rad9-AAD mutant by mutating both aromatic residues to alanine.
Analysis of the resulting rad9-AAD strain demonstrates no clear sensitivity to DNA damaging agents that create problems during S phase, including CPT, MMS and UV (Figure 7C) or in G2 to IR. However, some increased sensitivity is evident to CPT and MMS when the Rad4TopBP1 AAD mutant is present in the same strain. We next assayed the ability of rad9-AAD mutants to activate Chk1 in response to either IR or UV treatment. Consistent with the lack of sensitivity, the level of Chk1 phosphorylation after IR was not reduced (Figure 7D), either in rad9-AAD mutant alone or in the rad9-AAD rad4-Y599R double mutant when compared to the rad4-Y599R single. In response to UV treatment, there was again no decrease observed for the single rad9-AAD mutant, but a further and reproducible decrease was seen for the rad9-AAD rad4-Y559R double mutant when compared to rad4-Y599R alone (Figure 7E). Thus, the putative Rad9-AAD domain in S. pombe plays, at most, only a minor role in activating Rad3ATR in response to DNA damage and this is only revealed in the absence of the Rad4TopBP1 AAD.
Understanding the mechanism of ATR activation is an important facet of gaining insight into how cells respond to unwanted DNA structures, itself a key aspect in maintaining genomic integrity. TopBP1 was initially implicated in the ATR-dependent checkpoint in fission yeast and later this was extended to higher eukaryotes [53]. TopBP1 is a multi-BRCT-domain containing protein that acts to scaffold proteins during both the initiation of DNA replication and in response to DNA damage, a function dependent of the phospho-binding ability of the BRCT-domain pairs within TopBP1 [25]. In addition to scaffolding phospho-proteins, TopBP1 was shown to be able to directly activate ATR in Xenopus and human cells through a small domain of TopBP1 which is not part of any BRCT pair [17]. The ATR activating domain is sufficient, both in vitro and in vivo, to activate ATR - although it is not always necessary for ATR activation and the pathway in which this TopBP1 AAD domain functions is yet to be fully understood. It has recently been shown in S. cerevisiae that the AAD of the TopBP1 homolog (Dpb11) is also able to activate the ATR homolg (Mec1). However, the Dpb11 AAD plays a relatively minor role in checkpoint activation which is specific to G2 phase [24]. In S. cerevisiae, a second ATR activation domain within the C-terminal tail of the 9-1-1 subunit, Ddc1Rad9 acts to help activate ATR in G1 and G2 and it is only when the function of this AAD is ablated a role for the Dpb11 AAD becomes apparent [26]. However, loss of both domains does not prevent checkpoint activation entirely, suggesting other AADs or modes of activation. Conversely, in the Xenopus system, ATR activation via the TopBP1 AAD is evident in S phase.
Here we show that, in S. pombe, the activation of the ATR homolog (Rad3) by a Rad4TopBP1 AAD is conserved. We demonstrate that the Rad4TopBP1 AAD makes a contribution to checkpoint activation and that this is specific to G1/S phase and is not evident in G2. Note that log phase S. pombe spend little, if any, time in G1 and thus, while we can arrest cells before the onset of replication with cell cycle mutants, we cannot make a clear physiological distinction between G1 and S phase. We go on to demonstrate that, when DNA resection was limited in G2 by ablation of the Exo1 nuclease, checkpoint activation in response to DNA damage during G2 becomes partially dependent on the Rad4TopBP1 AAD, mimicking what we observed in G1/S cells. This leads us to propose that there is a threshold of ssDNA required for activation of the DNA damage checkpoint and that the Rad4TopBP1 AAD serves to amplify checkpoint signals when ssDNA is limiting.
We next used a genetic system to separate Rad3ATR activation from the production of DNA damage and therefore ssDNA, thus allowing us to assess the pathway of Rad3ATR activation dependent on the Rad4TopBP1 AAD. In this system, specific checkpoint proteins are recruited to a defined chromatin locus through dsDNA:protein binding [37], [39]. Interestingly, recruitment of any one of the three checkpoint proteins (Rad3ATR, Rad4TopBP1 and Rad9) tested was sufficient to generate a checkpoint response and these responses followed the expected dependencies. This suggests that the recruitment of multiple copies of a single checkpoint protein results in the formation of active checkpoint complexes that utilise the endogenous proteins. Using this system, we observed that the ability of the Rad4TopBP1 AAD to activate Rad3ATR is fully dependent on phosphorylation of H2A (γH2A) and requires the ability of Crb2 to bind γH2A. This leads us to conclude that, in the absence of ssDNA, the ATR activation domain of Rad4TopBP1 is particularly important for Rad3ATR activation and acts in a chromatin:protein interaction dependent manner. Taking these data together with the requirement of the Rad4TopBP1 AAD to amplify checkpoint signals in either G1/S or G2 when resection was limited, we propose that Rad4TopBP1 acts to amplify the checkpoint in a chromatin-dependent manner when single-stranded DNA levels are limiting. We can therefore hypothesise that there is a threshold level for the amount of active Rad3ATR required for a full checkpoint response. When ss-DNA is limited, such as in S-phase, the chromatin-dependent Rad4TopBP1 AAD-dependent pathway for Rad3ATR activation becomes important to amplify the levels of activated Rad3ATR to obtain a full checkpoint response (Figure 8).
In addition to analysing the Rad4TopBP1 AAD, we also created a mutant predicted to disable the Rad9 equivalent of the S. cerevisiae Ddc1Rad9 AAD and analysed the effect of this mutant in checkpoint activation. Unlike in S. cerevisiae, we observed no significant effect on DNA damage-induced checkpoint activation either in G1/S phase or G2. Although when combined with a Rad4TopBP1 AAD mutant, an additive effect to S-phase but not G2 DNA damage can be seen. This suggests that the Rad9 AAD acts in a separate but redundant pathway for Rad3ATR activation in G1/S with the Rad4TopBP1 AAD. It appears that, during evolution, the mechanism of activating the ATR pathway has diverged significantly with the roles of different ATR activating domains being of more or less importance in different organisms.
It will be interesting to establish if the ATR activating domain of TopBP1 in metazoan systems is particularly important in the context of low levels of ssDNA and whether its function is dependent on γH2AX, especially as a 53BP1(Crb2) and TopBP1 pathway for checkpoint activation in G1 has been previously reported in the mammalian system [54]. The differences in the dependencies of the specific ATR activators in different cell cycle phases between S. pombe and S. cerevisiae is not surprising as the checkpoint mechanism between these organisms has diverged. For example, in S. cerevisiae, the S phase checkpoint is activated independently of the 9-1-1 complex, whereas in S. pombe and mammalian cells ATR activation appears to be largely - if not entirely - dependent on 9-1-1 loading. Such distinctions are likely a result of evolutionary adaptation to the different cell cycle profiles of the two yeasts and it is interesting to note that significant evolutionary plasticity surrounds the interface between TopBP1 and the checkpoint apparatus. These distinctions will have to be considered when extrapolating mechanistic data from yeast to human systems. None the less, we believe that our findings shed light on the role of TopBP1 AAD in DNA damage responses and offer useful insights into metazoan mechanisms of DNA damage signalling.
Materials and Methods
S. pombe strain construction and biological methods
Standard S. pombe protocols were carried out as previously described [55]. rad4 and rad9 mutant strains were created using PCR site directed mutagenesis and integrated at their endogenous locus using Cre recombinase-mediated cassette exchange [56] In brief, this system uses a "base strain" which is engineered so that the gene of interest is either replaced with the ura4 marker (i.e. rad9), or in the case of essential genes (i.e. rad4) has the marker integrated immediately after the stop codon. In both cases the gene/marker and loci's promoter region are flanked by loxP and loxM sites. These two variant lox sites are incompatible with each other. The marker (and, for essential genes, the actual gene also) is then replaced by transforming in either the wild type (as a control: rad+) or the various mutated copies on a plasmid. These are flanked by the equivalent loxP and loxM sites and the plasmid expresses Cre recombinase, which results in loxP:loxP and loxM:loxM recombination. For cdc10-M17 synchronisation cells were grown to log phase at the permissive temperature (25°C) and shifted to the restrictive temperature of 36°C for 3.5 hours. Cells were then either irradiated with the indicated dose of gamma irradiation at 36°C and released at 25°C, or directly released at 25°C and irradiated at the given time points after release. cdc25-22 block and release [57] and lactose gradient synchronisation [12] were performed as described previously. For FACS analysis cells were resuspended in 50 mM tri-sodium citrate, 1 mg/ml final concentration RNAseA [Sigma], stained with 5 µg/ml Propidium iodide [Sigma] and analysed on FacsCalibur [Becton Dickinson].
For live cell imaging concentrated culture was mounted onto a 2.5% agar patch in standard YE medim [Microworks] and imaged on a Deltavsion Microscope. Septation index was counted as previously described [12]. lacO::NAT chk1-HA strains were created by inserting the 10 Kb lacO repeats into the PUC19 plasmid containing the NAT marker and homology to ura4. This was integrated into the genomic ura4 locus. The appropriate strains were transformed [58] with pRep41-GFP-LacI-NLS (GFP/LN) into which either rad3 or rad9 had been cloned in frame for N-terminal tagging or rad4 cloned in frame for C-terminal tagging. Transformants were grown and expression of the fusion protein induced by the removal of thiamine. All lacO repeats were checked by Southern hybridisation.
Biochemical assays
Protein extracts for western were prepared by TCA (trichloro-acetic acid) extraction from 1×108 cells and resuspended in SDS sample buffer [59]. Crude extracts for affinity analysis were prepared by mechanical disruption in liquid nitrogen. Antibodies used: α-HA [Santa cruz] 1∶2500, α-Myc [Santa cruz] 1∶2000, α-GFP [Roche] 1∶2500, α-H2ApS129 [Abcam] 1∶2500 or 1∶1000, α-Tubulin [Sigma] 1∶5000, α-Cdc2 Sc-53 [Santa cruz] 1∶2500. α-Cdc13 [Jacky Hayles] 1∶500. α-Cds1 1∶5000 [35]. The secondary antibodies used were Hrp rabbit α mouse [Dako] 1∶2500 or Hrp swine α Rabbit [Dako]1∶2500. Chk1-HA phosphorylation was quantified as a percentage of total signal minus back ground on a ImageQuant LAS 4000 [GE Healthcare]. Cds1 kinase assay was carried out as described [35].
Supporting Information
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PLOS Genetics
2012 Číslo 6
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