helicases are molecular motors that couple the energy of nucleoside triphosphate hydrolysis to the unwinding and remodeling of structured dna or rna [ 13 ] .
the number of helicases expressed in higher organisms is strikingly high , with approximately 1% of the genes in many eukaryotic genomes apparently encoding rna or dna helicases .
helicases are involved in virtually all aspects of nucleic acid metabolism , including replication , repair , recombination , transcription , chromosome segregation , and telomere maintenance [ 47 ] .
based on their substrates , helicases can be classified as dna or rna helicases , although some can function on both dna and rna molecules .
dna helicases have been reported to function in a variety of dna metabolic processes , including unwinding duplex or alternative dna structures ( triplex , g - quadruplex ) , stripping protein bound to dna , and chromatin remodeling [ 5 , 6 , 9 , 10 ] .
traditionally , helicases are known to unwind double - stranded dna or rna in an atp - dependent manner . however
, increasing evidence suggests that some helicases can rewind , or anneal , complementary strands of polynucleic acids in the presence or absence of nucleoside triphosphate ( figure 1 ) .
moreover , two so - called human helicases that were identified recently appear to only have atp - dependent rewinding activity [ 1115 ] .
these discoveries not only enrich the definition of helicases but also establish the presence of a new type of protein : annealing helicase .
the mechanism of this novel strand annealing activity and its biological consequences remain largely unknown . in this paper
, i will provide a brief overview of strand annealing activity found in various proteins across species and then focus on annealing helicases in humans and their potential mechanisms and functions .
most knowledge regarding the strand annealing activity of proteins has come from studies of model systems , including bacteria , yeast , and xenopus laevis .
the lehman lab reported the atp - dependent annealing activity of purified recombinant escherichia coli reca protein three decades ago .
although similar results were obtained by several other labs [ 1719 ] , the annealing activity of reca ( the human rad51 homolog ) is likely due to the binding of single - stranded dna ( ssdna ) , which forms a nucleoprotein filament .
the reca ( rad51 ) searches for homology along double - stranded dna ( dsdna ) and forms a stretching of dna duplex .
regulator protein stpa and recombination mediator protein reco  have been reported to promote annealing of complementary single stranded rna ( ssrna ) and ssdna , respectively [ 20 , 21 ] .
the dna replication polymerase ( dpo1 ) of sulfolobus solfataricus also possesses strand annealing activity . in yeast ,
the rothstein lab first found that rad52 protein binds ssdna and dsdna and promotes strand annealing .
later , the kowalczykowski lab demonstrated that rad51 and replication protein a ( rpa ) regulate rad52 's annealing function [ 2426 ] .
another yeast protein , rad59 , also stimulates complementary ssdna annealing [ 27 , 28 ] .
xenopus rna - binding protein x1rbpa was reported to promote rna  :  rna annealing .
for example , tat , one of six hiv regulatory proteins , encodes a small nuclear transcriptional activator and stimulates dna  :  dna annealing [ 30 , 31 ] ; dengue virus core protein also promotes rna  :  rna annealing .
the wilson lab first reported that rat heterogeneous nuclear ribonucleoprotein ( hnrnp ) a1 has strand annealing activity on both dna and rna , and the activity is inhibited by phosphorylation of kinase pka or pkc .
the annealing activity is restored by dephosphorylation of hnrnp by phosphatase 2a [ 34 , 35 ] .
mouse p53 protein inhibits the unwinding activity of t antigen helicase in vitro ; human p53 protein was also later found to inhibit several other helicases ' unwinding activity due to its strong dna  :  dna and rna  :  rna annealing activity .
the human rad51b , rad51c , rad51d , and xrcc2 protein complex catalyzes the strand - annealing reaction between a long linear ssdna ( 1.2  kb in length ) and its complementary circular ssdna . in summary , at least a dozen proteins , particularly those involved in dna replication and repair , have been demonstrated to possess strand annealing activity .
however , it is surprising to find that helicases , which unwind double stranded dna or rna , also possess strand annealing activity .
the annealing activity was first reported in rna helicases ( table 1 ) . in 1997 , the busch lab reported that human rh ii / gu rna helicase has rna folding activity  forming an intramolecular duplex . in 2001 , the stahl lab discovered that rna helicase p68 and its close relative p72 possess rna annealing activity for two complementary rna strands , and they also showed that ddx42p , another p68 homolog , has similar activity .
rna helicase a , also known as nuclear dna helicase ii or dhx9 , is a superfamily 2 ( sf2 ) helicase that unwinds dna - dna , rna - rna , and dna - rna strands with a 3-5 polarity .
recently , it was reported that rna helicase a promotes the annealing of trna3 , the primer for reversing transcription , to hiv-1 rna .
in addition to human rna helicases , some yeast and bacterial rna helicases also contain annealing ability .
for example , the jankowsky lab demonstrated that ded1 from saccharomyces cerevisiae , in addition to its rna unwinding activity , facilitates the formation of rna duplexes .
the lambowitz lab found that mss116p of s. cerevisiae promotes the annealing of the oligonucleotides in the absence of atp .
crhr , a cyanobacterial rna helicase , was also found to promote duplex formation in the presence of atp .
compared with rna helicase , more dna helicases have been found to possess annealing activity ( table 1 ) .
the janscak lab is the first to report that both unwinding and annealing activity resided in a dna helicase , recq5 in 2004 .
the janscak lab not only discovered the novel activity of recq5 , but also mapped the unique c - terminal portion ( aa 411991 ) that possesses the dna strand - annealing activity .
this was the first demonstration of a dna helicase with an intrinsic dna annealing function residing in a separate domain of the same polypeptide ( figure 2 ) .
subsequently , many helicases , particularly recq family helicases , have been found to possess annealing activity . in humans , there are five recq homologs , and mutations in three of these genes ( blm , wrn , and recq4 ) are associated with bloom 's , werner , and rothmund - thomson syndromes , respectively .
although no disease has been linked to mutations of recq5 , recq5 mice are highly cancer prone and display genomic instability [ 60 , 61 ] .
a single nucleotide polymorphism in recq1 correlates with decreased survival of pancreatic cancer patients [ 62 , 63 ] .
annealing activity was reported for the bloom 's syndrome helicase , blm , in 2005 by two independent groups [ 51 , 53 ] .
recently , the brill lab further identified a subdomain of the blm / sgs1 n - terminus that contains annealing activity in vitro .
not only do human blm and yeast sgs1 display annealing activity , so does the drosophila blm homolog ( mus309/dmblm ) [ 51 , 64 , 65 ] .
the wrn helicase was reported to contain strand pairing activity , and the annealing activity was mapped to the c - terminal region ( aa 10721150 ) .
later , the vindigni lab reported that recq1 efficiently promotes strand annealing as a higher order oligomer ( pentamer or hexamer ) , while smaller oligomeric states ( dimer or monomer ) act to unwind duplex dna .
different quaternary states of recq1 , modulated by binding of ssdna and atp , are associated with its strand annealing or unwinding activity ( discussed later ) . unlike other recq helicases , neither endogenous recq4 purified from hela cell extracts   nor recombinant recq4 purified from e. coli   originally showed unwinding activity
. the unwinding activity of recq4 appears to have been masked by its strong intrinsic dna annealing activity [ 54 , 69 ] . because of its robust annealing activity
, the presence of a third strand ( e.g. , same sequence as the labelled oligo but unlabelled ) is required to demonstrate unwinding by recq4 .
furthermore , the n - terminus ( aa 1492 ) is required for the annealing activity of recq4 .
although it was later reported that recq4 could displace short oligonucleotides ( 22  mer ) without a third strand , the fact that longer oligonucleotides ( 30  mer ) require a third strand [ 69 , 70 ] indicates the strong annealing activity resides in the recq4 peptide .
in addition to the recq helicase family , several other dna helicases possess strand annealing activity too ( table 1 ) .
the pif1 helicase family is a group of 5  3 directed , atp - dependent , super - family ib helicase that is found in nearly all eukaryotes .
the purified recombinant human pif1 proteins display robust annealing activity without atp , and this activity resides in the n - terminal region ( aa 1180 ) of the protein   ( figure 2 ) .
dna2 is a helicase and nuclease involved in okazaki fragment processing , double - strand break ( dsb ) repair , telomere regulation , and mitochondrial function .
point mutation analysis revealed that the annealing activity does not require either the nuclease or the helicase activity of dna2 , indicating that the rewinding activity is uncoupled from nuclease and/or unwinding activity .
mutations in csb gene cause cockayne syndrome , a rare inherited genetic disorder characterized by uv sensitivity , severe neurological abnormalities , and progeroid symptoms .
the csb protein catalyzes strand annealing of complementary ssdna , while both atp and rpa inhibit its annealing activity .
twinkle is a human mitochondrial dna helicase that is associated with heritable neuromuscular diseases , and it has ntp - independent annealing activity .
the t4 phage uvsw helicase   two archaea helicases ( hjm / hel308a   and hel112 ) , and mycobacterium tuberculosis xpb   also contain strand annealing activities .
the term  annealing helicase  was created when two helicase domain - containing proteins were discovered to possess annealing activity and no unwinding activity . in 2008 , the kadonaga lab discovered that harp is an atp - dependent annealing helicase .
harp ( hepa - related protein ) , also known as smarcal1 ( swi / snf - related , matrix - associated , actin - dependent regulator of chromatin , subfamily a - like 1 ) , is a member of the swi / snf family protein .
mutations in harp are associated with schimke immuno - osseous dysplasia ( siod ) , a multisystem autosomal recessive disorder characterized by short stature , kidney disease , and a weakened immune system .
the majority of siod patients have t - cell deficiency and associated risk for opportunistic infection , a common cause of death .
loss of harp affects cellular proliferation and differentiation , and the response to replication stress . following the kadonaga lab 's report
, three other labs found similar enzymatic activity for harp and further defined its biological functions [ 1113 ] .
harp binds directly to rpa via a conserved n - terminal motif and anneals rpa - coated complementary ssdna .
it was proposed that harp might dictate its role in the s - phase - specific dna damage response to protect stalled replication forks by minimizing the accumulation of ssdna regions and facilitating the repair of collapsed replication forks [ 1113 , 80 ] .
the chen lab further identified that two hp domains , each has about 60 residues , dictate the annealing activity of harp .
more recently , the cortez lab reported that the first hp domain is not required for annealing activity , and , intriguingly , harp is able to catalyze branch migration of holliday junctions ( hjs ) and regression of replication forks .   after discovering the unique activity of the harp protein ,
the kadonaga lab identified another annealing helicase that they named annealing helicase 2 ( ah2 ) .
ah2 was previously named zranb3 ( zinc - finger , ran - binding domain containing 3 ) , a member of the snf2 family .
similar to harp , the purified recombinant ah2 protein displays dna - dependent atpase activity and atp - dependent rewinding activity .
however , unlike harp , ah2 lacks a conserved rpa - binding domain and does not interact with rpa .
in addition , ah2 contains an hnh motif at its extreme c - terminus ( figure 2 ) , which is common in prokaryotes and is often associated with nuclease activity .
contrary to expectations , the purified recombinant ah2 protein does not exhibit nuclease activity . with no disease linked to ah2 or genetic model generated for ah2 ,
interestingly , the direction of both annealing helicases ( harp and ah2 ) has not been examined in vitro . since classic dna - dependent atpases that are bonafide helicases that have the ability to catalytically separate complementary strands behave in a directional manner with respect to the strand that the helicase protein is presumed to be bound , depend on which we define 3-5 or 5-3 helicase , therefore
, it would be of interest to know whether harp or ah2 translocate in a directionally specific manner .
specific helicases need to function on the appropriate nucleic acid substrate at the appropriate time .
in addition , these enzymes might be required to facilitate unwinding and/or rewinding under different circumstances .
indeed , several bloom 's syndrome missense mutant proteins lack unwinding activity , but still possess strand annealing activity that is even greater than wild type blm , indicating that the misregulation of unwinding and rewinding activity may be one of the pathogenic factors .
a key emerging question is how these two opposite activities of helicase are precisely regulated .
the number of helicase motifs varies from seven , nine , to twelve , which are responsible for nucleic acid binding , ntp hydrolyzing , and dna or rna unwinding [ 2 , 87 ] .
some helicases also contain accessory domain(s ) at the n- or c - terminus , such as nuclease domain and various protein - protein interaction domains . as shown in figure 2 , studies of human blm helicase and its orthologs including budding yeast sgs1 and drosophila blm revealed that its n - terminal region contains strand annealing activity .
studies of the recq5 helicase revealed that the c - terminus is responsible for its annealing activity .
furthermore , studies of recq4 protein revealed that some missense mutants lose unwinding activity but still possess strand annealing activity .
these results suggest that the dna unwinding and strand annealing activities can be uncoupled , but the question remains whether there is a conserved domain that controls annealing activity .
the cortez lab found that only the second hp domain is required for the annealing activity of harp   ( figure 2 ) .
alignment of the hp domain with ah2 reveals a putative  hp - like  domain in the ah2 protein ( residues 712820 ) .
although hp domain - like amino acids are found in the ah2 , it is unlikely that the hp domain is a universal element that governs annealing activity across helicases .
for example , the n - terminal region ( residue 156 ) of recq1   and the c - terminal region ( aa 10721150 ) of the wrn helicase   are required for their respective annealing activities .
alignment of the  annealing domains  of these two recq helicases with the hp domains present in harp and the hp - like domain in ah2 reveals no significant conserved residue .
although the n - terminal domain of recq4 and the c - terminal region of recq5 share certain identity and similarity , alignment of the n - terminus of recq4 and blm , and the c - terminus of recq5 does not result in any significant homology ( data not shown ) .
thus , it is unlikely that a single conserved domain is responsible for the annealing activity of these helicases .
certain helicases may self - assemble to form dimers or higher order oligomers , and this can influence their catalytic activity or biological function [ 13 ] .
human recq1 helicase efficiently promotes strand annealing as a higher order oligomer ( tetramer ) while smaller oligomeric states ( dimer or monomer ) acting to separate duplex dna [ 50 , 88 ] .
electron microscopy reconstructions of the higher order oligomeric form revealed that a cage - like structure forms a hollow channel , which may facilitate the annealing activity of recq1 . both consistent and inconsistent with the findings of recq1 , hel112 , a homologue of human recq5 in sulfolobus solfataricus , exists as two predominant stable oligomeric states : monomer and dimer .
only the monomeric form has 3-5 dna - helicase activity , while both the monomer and the dimer possess strand - annealing activity .
these findings raise the possibility that higher order oligomers promote annealing activity and smaller order oligomers promote unwinding activity .
nevertheless , additional studies are needed to address the relationship between the oligomerization and dual activities of helicases . for helicases that possess unwinding activity
, it makes sense that atp fuels the unwinding activity , in turn , inhibits the annealing activity .
indeed , this has been seen in dna helicases such as blm , pif1 , as well as the e. coli cas3 helicase .
it was also reported that , for the rna helicase ddx42p , atp triggers rna strand separation while adp triggers annealing of complementary rna strands . however , for helicases with no detectable unwinding activity , it is largely unknown how atp regulates their annealing activity .
for example , how atp inhibits csb 's annealing activity , while harp and ah2 require atp [ 14 , 15 ] . rather than fueling the unwinding activity by hydrolyzing atp
for example , the strand - annealing activity of recq5 is strongly inhibited by atps , a poorly hydrolyzable analog of atp .
this effect is alleviated by mutations in the atp - binding motif of recq5 , indicating that the atp - bound form of the protein can not promote strand annealing .
atps also inhibits the annealing activity of blm , wrn , and crhr helicases . for recq1
, atp binding induces a conformational change in the protein that serves as a molecular switch from a strand - annealing to a dna - unwinding mode . if atp indeed functions as a switch to regulate or balance the unwinding and rewinding activity of helicases , a promising avenue for future research will be to investigate the regulation mechanism , for example , structural determination of helicases with and without atp .  
rpa is a fundamental protein involved in all aspects of cellular metabolism ( see review [ 90 , 91 ] ) . in dna repair processes ,
rpa physically coats ssdna to protect it from degradation by nucleases and also serves as a scaffold protein to recruit other repair proteins ( e.g. , rad51 ) to strand break sites . at the same time
, rpa promotes checkpoint signaling after replication fork stalling through activation of atratrip and rad17 complexes .
rpa has been shown to stimulate the unwinding activity of many helicases in vitro , including wrn [ 92 , 93 ] , blm , recq1 , and fancj . in turn , the strand annealing activity of recq1 , pif1 , and csb   is inhibited by rpa .
rpa depletion causes a dramatic reduction in the formation of the annealing products in xenopus egg extracts , suggesting that rpa is required for single - strand annealing .
the annealing helicase harp associates with rpa , but whether it is just a physical interaction or if rpa modulates harp 's annealing function remains unknown .
for ah2 , yet to be identified proteins may regulate its nuclease and/or annealing activity .
in addition to rpa , several other proteins have been shown to promote annealing activity of helicases .
the 65  kda subunit of u2af is reported to mediate the annealing of complementary single - stranded rna or single - stranded dna , which reverses the action of rna helicase a .
the purified human p53 protein inhibits the unwinding activity of several dna helicases , such as t antigen dna helicase , dna helicases i and ii of e. coli , and rna helicases p68 , by promoting the rapid renaturation of complementary strands .
the endonuclease xpg is also reported to stimulate the annealing activity of the wrn helicase .  
catalytic activities of proteins can be modulated by posttranslational modifications , such as phosphorylation / dephosphorylation , acetylation , ubiquitination , and sumoylation .
for example , endogenous recq1 is phosphorylated upon treatment of cells with ionizing radiation ( ir ) , ultraviolet ( uv ) , and hydroxyurea ( hu ) ; blm is phosphorylated by atm in response to ir [ 101 , 102 ] , atr in response to hu , and several other kinases  ck1 , cdc2 , and mps1 .
wrn is phosphorylated by atm and atr in response to replication fork arrest [ 107 , 108 ] .
harp is phosphorylated by atm , atr , and dna - pk in response to the dna damage checkpoint [ 11 , 81 ] .
indeed , the strand annealing activity of csb is increased by dephosphorylation with phosphatase i and decreased by phosphorylation with ckii .
thus , helicase function may in specific cases be regulated by post translational modification through modulation of its strand annealing activity .
nevertheless , it remains largely unknown how annealing activity is modulated by these protein modifications .
the biological function of so - called real helicases , which possess unwinding activity that includes recq family helicases [ 4 , 6 ] , pif1 , and dna2 , has been extensively reviewed .
the physiological relevance of helicase annealing activity is revealed by the finding that several mutations observed in siod patients result in defective annealing activity in harp protein [ 14 , 78 , 111 ] .
although a limited number of annealing helicases have been identified , several biological functions have been indicated by related experimental evidence .
stalled replication forks can arise during normal chromosome replication or in the presence of dna lesions , but will collapse if being left unrepaired due to the presence of long stretches of ssdna .
annealing helicases might stabilize stalled replication forks through pairing the parental ssdna , migrating chicken foot / holliday junctions structures , and/or directly participating in repair of the lesion ( figures 3(a ) and 3(b ) ) .
green fluorescent protein ( gfp)-tagged harp is recruited to stalled replication forks . compared with ssdna and dsdna , fork dna is a preferred substrate for ah2 binding and atpase activity , indicating that the forked dna is a physical substrate for ah2 .
because harp and ah2 can act as an opposing force to unwinding activities in vivo , there are obvious potential implications for the role of harp and ah2 in dna replication and dna repair activities ( discussed below ) .
harp and ah2 might dictate their role in protecting stalled replication forks by minimizing the accumulation of ssdna regions and facilitating the repair of collapsed replication forks during dna replication .
consistent with its function on ssdna , four research groups have found that harp associates with the rpa complex [ 1113 , 81 ] , which possibly anchors harp to the ssdna .
cells depleted of harp accumulate ssdna and display increased sensitivity to aphidicolin and hu [ 12 , 13 ] .
dna fibre analyses show that restart of replication forks after 2 hours of aphidicolin treatment is reduced in harp - depleted cells [ 11 , 81 ] .
these data suggest that harp promotes fork stability and restart by reannealing long stretches of ssdna generated at stalled replication forks .
indeed , very recently , the cortez lab demonstrated that in vitro harp can bind and branch - migrate three - way and four - way dna structures , and catalyze extensive fork regression of model replication forks .  
the recq family helicases have been well recognized to function in damaged replication forks . wrn-
deficient cells are hypersensitive to replication blocking agents , including hu [ 107 , 114 ] and dna - interstrand cross - linking drugs .
blm - deficient cells are also hypersensitive to hu   and mitomycin c ( mmc ) . although less sensitive to hu as blm - deficient and wrn- deficient cells , recq4-deficient cells are sensitive to hu . recq1-depleted human cells are sensitive to hu   and camptothecin ( cpt ) .
in addition , it has been reported that both blm and wrn are recruited to blocked replication forks in vivo   and can catalyze fork regression in vitro [ 120 , 121 ] . moreover
, the orren lab recently demonstrated that wrn and blm reestablish functional replication forks to overcome fork blockage .
all these evidence suggest that recq helicase , particularly blm and wrn , participate in remodeling of stalled replication forks .
however , it remains unknown how these helicases exert their annealing activity to contribute to replication fork restart .
in addition to the recq helicases , human pif1 helicase specifically recognizes and unwinds dna structures resembling putative stalled replication forks . using yeast ribosomal dna as a dna replication model , it has been shown that the events of replication fork block are increased in dna2 mutants .
in particular , dna2 is involved in okazaki fragment processing , and it will be of interest to determine if its annealing activity stabilizes the lagging strand .
unlike harp and ah2 , it would seem most likely that proteins with both unwinding and annealing activities might coordinate those activities to catalyze strand exchange and/or branch migration , such as generating and migrating a holliday junction or chicken foot structure for stalled dna replication forks ( figure 3(a ) ) . the function of the recq helicase family in dna repair processes is well recognized .
the csb protein is known to function in transcription - coupled dna repair ( tcr ) .
cells with harp depletion display a higher frequency of dsb [ 13 , 81 ] and sensitivity to dna - damage ( e.g. , ir , mmc , and cpt ) .
harp is phosphorylated by atm , atr , and dna - pk in vitro , and the mobility of harp in sds gels is altered when it is isolated from cells treated with dna damage ( hu , ir , and uv radiation ) .
taken together , evidence suggested that annealing helicases are involved in dna repair , but the question remains how the annealing activity contributes to dna repair processes .
of the various types of dna damage , dsb may be the most common and cytotoxic to cells .
dsbs are repaired by non - homologous end joining ( nhej ) , homologous recombination ( hr ) , and microhomology - mediated end joining ( mmej ) .
all three dna repair pathways need pairing of complementary single - stranded dna , particularly hr and mmej .
there are mainly four forms of hr in higher eukaryotic cells : double holliday junctions ( dhj ) formed through crossover , synthesis - dependent strand annealing ( sdsa ) utilized by non - crossover , single - strand annealing ( ssa ) pathway between two repeat sequences , and break - induced replication ( bir ) pathway that repairs 
hr pathways are initiated by rad51 that searches and invades base - paired strands of homologous dna molecules , then d - loop and hjs are formed consequently ( figure 3(b ) , left ) .
recq helicases have strand exchange activity [ 51 , 126 ] and hjs branch migration ability [ 120 , 121 ] . in the dhj pathway
, it is very likely that the recq helicases , particularly wrn and blm , coordinate their unwinding and annealing activities to separate intact double - stranded dna and pair invading strand to template in the early stage , migrate hjs , and finally help to cleave hjs .
sdsa is a mechanism in which homology - mediated repair of dsbs occurs without formation and resolution of ligated hjs ; it anneals the newly synthesized strand with the single strand resulting from resection of the second end ( figure 3(b ) , left ) .
mmej or ssa use microhomologous sequences ( 525  bp ) or long homologies ( > 30  bp ) to align broken ends before ligation ( figure 3(b ) , right ) .
nevertheless , dhj , sdsa , mmej , and ssa pathways heavily rely on single - strand dna - annealing activity driven by proteins . on the other hand ,
dna helicases are recognized to play negative regulatory roles in recombination / repair through antirecombination , by disrupting presynaptic filaments prior to strand invasion , or by resolving d - loops before they can be extended and converted into replication forks .
although annealing helicases harp and ah2 have not been investigated in the dna repair pathways discussed above , both recq4 and blm helicases are reported to be required to promote sdsa [ 128 , 129 ] . in the mmej or ssa pathways ,
nuclease activity is required in the flap - trimming step ( figure 3(b ) , right ) .
in fact , it has been reported that several helicases , including annealing activity containing helicase recq5 , blm , and wrn , stimulate cleavage activity of flap endonuclease 1 ( fen-1 ) .   besides their function in dna replication and repair , another function of annealing helicases might be in dna transcription ( figure 3(c ) ) .
cas3 is a superfamily 2 helicase that possesses atpase , helicase , and nuclease activities as evident in the cas3 protein of streptococcus thermophilus .
recently , the e. coli cas3 protein was found to promote r - loop ( rna  :  dna hybrid ) formation within duplex dna in the absence of atp and to disassemble r - loops in the presence of atp , which is more relevant to structures formed in dna transcription .
given the fact that the csb protein is recruited to the site of a chromatin bound uv - stalled rna polymerase ii complex   and that the recq5 helicase physically and functionally associates with rna polymerase ii [ 135138 ] , it will be of interest to determine if the csb and recq5 use their annealing activity to stabilize the transcription machine as a dna damage response . a number of recq helicases are implicated in telomere metabolism , including wrn , blm , and recq4 .
the purified recombinant pif1 proteins bind telomeric dna with a 100-fold higher affinity compared to random sequence , and telomere shortening was observed when pif1 was overexpressed .
telomere effects of dna2 proteins have been reported in s.  cerevisiae and s.  pombe [ 144 , 145 ] .
it will be of interest to know how these helicases exert their strand annealing activity , in particular recq4 that has robust annealing activity , to function in telomere maintenance .
annealing helicases might participate in telomere metabolism , where single strand overhang could require an annealing helicase to form a more stable structure , such as t - loop ( figure 3(d ) ) . for cells which operated by the alternative
lengthening of telomere ( alt ) pathway , it is possible that strand annealing / strand exchange catalyzed by certain recq helicases may participate in telomere - sister chromatid exchange and hr - dependent dna replication ( figure 3(d ) ) .
coordination of strand annealing and unwinding activity at the g - rich telomeric end may influence telomere stability by affecting dna replication and repair processes , such as resolving g4 dna .
the characteristic feature of this class of enzymes is that they contain a conserved atpase domain with the seven classic helicase - related motifs .
the swi2/snf2 atpases are grouped within the sf2 helicase superfamily , and they have a distinct primary sequence signature defined by the spacing between helicase motifs iii and iv as well as conserved features of their sequences within the domain .
although most swi2/snf2 atpases do not have duplex dna strand separating activity , they retain other features of helicases , including directional dna translocation fueled by atp hydrolysis , participation in chromatin structure modeling , regulation transcription , and dna repair .
unlike harp and ah2 , not all helicase domains containing snf2 family proteins have annealing activity , but like harp and ah2 , many of them have been identified to promote branch migration of hjs in an atp hydrolysis - dependent manner . in prokaryotes ,
ruvab and recg have been shown to promote branch migration of hjs [ 147 , 148 ] . in eukaryotes ,
several members of snf2 can promote fork regression and/or branch migration of hjs , including rad54 , rad5 [ 150 , 151 ] , and hltf .
chd7 , a helicase domain containing snf2 protein accounted for the majority of charge syndrome , plays a role in transcription regulation by chromatin remodeling .
although fancm is not a snf2 family member , it consists of an n - terminal sf2 helicase domain and a c - terminal inactive endonuclease domain .
fancm is able to branch migrate hjs and replication forks in vitro [ 154 , 155 ] .
very recently , several helicase domain containing snf2 proteins , including smarca2 and smarca4 , were identified to cause nicolaides - baraitser syndrome   and coffin - siris syndrome .
interestingly , most of the patient mutations are located in the helicase domain , suggesting its importance for their function .
however , it remains unknown whether they have strand annealing activity in vitro . in conclusion , it is a growing family of proteins that contain atpase / helicase domain , which use their enzymatic activity to regulate chromatin structure and gene expression .
the discovery of annealing helicases establishes the presence of a class of enzymes that possess only rewinding activity and opens a new area of research . the range of proteins that function as annealing helicases remains to be determined .
researchers now hope to determine the biological function of harp and ah2 more fully , as well as to discover more of these types of enzymes .
annealing helicases could also potentially be found for rna - dna and rna - rna hybrids , expanding the research into areas such as protein synthesis , rna stability , and gene silencing .
the coordinated action of unwinding and annealing may play a role in fork regression or synthesis - dependent strand annealing , in the pathway for dsb repair , as well as in transcription and telomere metabolism .
the challenge will then be to understand how cells regulate helicase unwinding and rewinding activity in vivo , and determine where or when the annealing activity of helicase is needed . from an experimental standpoint , it would be great interest to identify and characterize separation of function mutants which : ( 1 ) inactivate helicase activity but retain strand annealing ; ( 2 ) inactivate strand annealing but retain helicase activity . characterization of clinically relevant or engineered helicase core domain and auxiliary domain missense mutants may be valuable to determine which biological pathways / steps require strand annealing versus unwinding activity .
finally , a better understanding of the biological function of annealing helicases is likely to provide the basis for treating a variety of human disorders , such as siod of harp , premature aging of recq helicases , and cancers .
 helicases are enzymes that use atp - driven motor force to unwind double - stranded dna or rna . recently 
 , increasing evidence demonstrates that some helicases also possess rewinding activity  in other words , they can anneal two complementary single - stranded nucleic acids . 
 all five members of the human recq helicase family , helicase pif1 , mitochondrial helicase twinkle , and helicase / nuclease dna2 have been shown to possess strand - annealing activity . moreover , two recently identified helicases  harp and ah2 have only atp - dependent rewinding activity . 
 these findings not only enhance our understanding of helicase enzymes but also establish the presence of a new type of protein : annealing helicases . this paper discusses what is known about these helicases , focusing on their biochemical activity to zip and unzip double - stranded dna and/or rna , their possible regulation mechanisms , and biological functions . 