Separable recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation

During eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, but find that the absence of the replisome adaptor protein Ctf4 only impairs lagging-strand initiation. Our results suggest that distinct modes of Pol α recruitment exist for replication initiation and elongation, and are consistent with distributive association of Pol α with the replisome. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α, and demonstrate that this checkpoint requirement is due to impaired origin firing as opposed to reduced lagging-strand priming. Author summary Half of each eukaryotic genome is replicated continuously as the leading strand, while the other half is synthesized discontinuously as Okazaki fragments on the lagging strand. The bulk of DNA replication is completed by DNA polymerases ε and δ on the leading and lagging strand respectively, while synthesis on each strand is initiated by DNA polymerase α-primase (Pol α). Using the model eukaryote S. cerevisiae, we modulate cellular levels of Pol α and interrogate the impact of this perturbation on both replication initiation on DNA synthesis and cellular viability. We observe that Pol α associates dynamically at the replication fork for initiation on both strands. Although the initiation of both strands is widely thought to be mechanistically similar, we determine that Ctf4, a hub that connects proteins to the replication fork, specifically recruits Pol α to the lagging strand but is not required for leading-strand initiation. We also find that decreased leading-strand initiation results in a checkpoint response that is necessary for viability when Pol α is limiting. Because the DNA replication machinery is highly conserved from budding yeast to humans, this research provides insights into how DNA replication is accomplished throughout eukaryotes.

the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki 23 fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We 24 observe that both Okazaki fragment initiation and the productive firing of replication origins are 25 sensitive to Pol α abundance, but find that the absence of the replisome adaptor protein Ctf4 26 only impairs lagging-strand initiation. Our results suggest that distinct modes of Pol α 27 recruitment exist for replication initiation and elongation, and are consistent with distributive 28 association of Pol ⍺ with the replisome. Additionally, we observe that activation of the 29 checkpoint becomes essential for viability upon severe depletion of Pol α, and demonstrate that 30 this checkpoint requirement is due to impaired origin firing as opposed to reduced lagging-31 strand priming.  (Villa et al., 2016). At least some of these roles 100 for Ctf4 are independent of Pol α binding. The metazoan Ctf4 ortholog, AND-1 also stimulates 101 Pol α binding to chromatin, and is required for efficient DNA replication (Zhu et al., 2007).  asynchronous population are in G1, G2 or M phase, we compared wild-type POL1 expression 134 during S phase to 0.5% and 0.05% galactose in our inducible system at the zero and 60 minute 135 timepoints after release from alpha-factor-mediated G1 arrest (Fig. S1A). We estimate that the 136 expression level at 0.05% galactose is slightly lower than endogenous during the relevant phase 137 of the cell cycle, consistent with several phenotypes described below.

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To analyze Okazaki fragment biogenesis, we crossed the GDPOL1 allele into a strain Okazaki fragments was possible after 1h rapamycin treatment: therefore, all Okazaki fragment 145 labeling and sequencing experiments were conducted after a 4h sugar switch to reduce Pol α 146 levels, followed by 1h ligase depletion by rapamycin (Fig. 1B).

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By end-labeling unligated Okazaki fragments, we observed that cells with wild-type POL1 did Okazaki fragment length profile was shifted slightly upwards such that fragments were clearly 155 still phased by nucleosomes, while lower galactose concentrations (0.005%) showed a 156 significant loss of signal (Fig. 1D cf. lanes 5&6). To confirm that this loss of signal reflected a 157 further length increase (and therefore a reduction in the number of ends being labeled), we 158 analyzed Okazaki fragments by Southern blot using a whole-genome probe: as anticipated for 159 severely perturbed lagging-strand priming, Okazaki fragments at 0.005% galactose were 160 significantly larger than at 0.014% (Fig. 1E). We conclude that limiting levels of Pol α lead to 161 reduced priming frequency on the lagging strand, resulting in longer Okazaki fragments, and 162 that S. cerevisiae cells can sustain growth when Okazaki fragment length is substantially

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To analyze the location of Okazaki fragment termini, we purified and sequenced Okazaki 170 fragments (Smith and Whitehouse, 2012) from wild-type and GDPOL1 strains shifted to low 171 galactose concentrations for 4h before 1h ligase depletion. Galactose concentration does not 172 significantly affect the distribution of Okazaki fragment termini in wild-type cells (Fig. 1F).

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However, we observed that both the 5' (

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Interestingly, we observed that the efficiency of productive replication origin firing is impaired at 201 similar Pol1 concentrations (0.023% galactose) to those that impair Okazaki fragment initiation 202 (0.014% galactose) (Fig 1D&E). This dramatic decrease in origin efficiency likely contributes to

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In the absence of Ctf4, Okazaki fragments increase in length at ~0.06% galactose (Fig. 3A,)-242 significantly higher than the 0.014% observed for a CTF4 wild-type strain (cf. Fig. 1D&E). The  showed normal origin efficiency down to 0.023% galactose, followed by a decrease at 0.014% a 262 further reduction at 0.005% galactose (Fig. 3E). Because origin firing efficiency at low Pol α is 263 either unaffected or very slightly increased by the absence of Ctf4-Pol α interaction (cf.

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To determine whether the requirement for checkpoint activation was due to deregulated lagging-346 strand priming or impaired leading-strand initiation, we compared the growth of 347 GDPOL1;mec1Δ;sml1Δ strains with and without CTF4 or pol1-4A at 0.5% and 0.05% galactose.

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Loss of Ctf4-Pol α interactions affects Okazaki fragment initiation at relatively high Pol α 349 concentrations, before leading-strand initiation is impaired. Thus, ctf4Δ and pol1-4A are 350 effectively separation of function mutants: at 0.05% galactose, Okazaki fragment initiation is 351 reduced by the absence of Ctf4-mediated recruitment of Pol α while leading-strand initiation is 352 not (Fig. 3). GDPOL1;mec1Δ;sml1Δ;ctf4Δ cells grow slowly relative to MEC1 or CTF4 cells: 353 however, growth was minimally affected by galactose concentrations (Fig. 4E). This result is 354 recapitulated with GDpol1-4A;mec1Δ;sml1Δ strain, which showed essentially no growth defect 355 at either Pol α concentration (Fig. 4E). Therefore, perturbed lagging-strand synthesis does not 356 directly cause growth defects the absence of a functional checkpoint. We conclude that cells

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Following the sugar switch methods above, Okazaki fragments were accumulated by adding 486 rapamycin (Spectrum 41810000-2) to 1ug/mL to anchor away Cdc9, which is tagging with FRB, 487 for 1h (Haruki et al., 2008). These cells were collected through centrifugation and immediately 488 processed or stored at -80°C. Genomic preps from spheroplasts were completed as previously

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For southern blot analysis, unlabeled genomic DNA was normalized to total genomic DNA, and 497 run on 1.3% denaturing agarose gels. After electrophoresis, the gel was blotted onto a 498 nitrocellulose membrane (Fisher 45-000-932) overnight. Next the membrane was crosslinked 499 and washed, then hybridized overnight with a probe synthesized with random hexamers labeling 500 kit (Fisher 18187-013) and sheared genomic DNA. After two low stringency washes, 501 membranes were dried then exposed to phosphor screens.

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Okazaki fragment purification, sequencing, and analysis 504 Genomic DNA was boiled at 95°C for 5 min then salt was added to 300mM NaCl, pH 12.

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The single-strand Okazaki fragments were boiled at 95°C for 5 min and cooled quickly on ice