Pif1, RPA, and FEN1 modulate the ability of DNA polymerase δ to overcome protein barriers during DNA synthesis
2020; Elsevier BV; Volume: 295; Issue: 47 Linguagem: Inglês
10.1074/jbc.ra120.015699
ISSN1083-351X
AutoresMelanie Sparks, Peter Burgers, Roberto Galletto,
Tópico(s)Signaling Pathways in Disease
ResumoSuccessful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication. Successful DNA replication requires carefully regulated mechanisms to overcome numerous obstacles that naturally occur throughout chromosomal DNA. Scattered across the genome are tightly bound proteins, such as transcription factors and nucleosomes, that are necessary for cell function, but that also have the potential to impede timely DNA replication. Using biochemically reconstituted systems, we show that two transcription factors, yeast Reb1 and Tbf1, and a tightly positioned nucleosome, are strong blocks to the strand displacement DNA synthesis activity of DNA polymerase δ. Although the block imparted by Tbf1 can be overcome by the DNA-binding activity of the single-stranded DNA-binding protein RPA, efficient DNA replication through either a Reb1 or a nucleosome block occurs only in the presence of the 5'-3' DNA helicase Pif1. The Pif1-dependent stimulation of DNA synthesis across strong protein barriers may be beneficial during break-induced replication where barriers are expected to pose a problem to efficient DNA bubble migration. However, in the context of lagging strand DNA synthesis, the efficient disruption of a nucleosome barrier by Pif1 could lead to the futile re-replication of newly synthetized DNA. In the presence of FEN1 endonuclease, the major driver of nick translation during lagging strand replication, Pif1-dependent stimulation of DNA synthesis through a nucleosome or Reb1 barrier is prevented. By cleaving the short 5' tails generated during strand displacement, FEN1 eliminates the entry point for Pif1. We propose that this activity would protect the cell from potential DNA re-replication caused by unwarranted Pif1 interference during lagging strand replication. Impeded DNA replication can lead to replication fork stalling and, potentially, genomic instability (1Patel D.R. Weiss R.S. A tough row to hoe: when replication forks encounter DNA damage.Biochem. Soc. Trans. 2018; 46 (30514768): 1643-165110.1042/BST20180308Crossref PubMed Scopus (12) Google Scholar, 2Gaillard H. García-Muse T. Aguilera A. Replication stress and cancer.Nat. Rev. Cancer. 2015; 15 (25907220): 276-28910.1038/nrc3916Crossref PubMed Scopus (397) Google Scholar). Yet many barriers must be overcome during DNA replication, such as DNA secondary structures and tightly bound proteins. During DNA replication, nucleosomes are recycled from the parental strand and redeposited randomly on the two daughter strands (3Groth A. Replicating chromatin: a tale of histones.Biochem. Cell Biol. 2009; 87 (19234523): 51-6310.1139/O08-102Crossref PubMed Scopus (20) Google Scholar, 4Annunziato A.T. The fork in the road: histone partitioning during DNA replication.Genes. 2015; 6 (26110314): 353-37110.3390/genes6020353Crossref PubMed Scopus (33) Google Scholar, 5Prado F. Maya D. Regulation of replication fork advance and stability by nucleosome assembly.Genes. 2017; 8: 4910.3390/genes8020049Crossref Scopus (19) Google Scholar, 6Gan H. Serra-Cardona A. Hua X. Zhou H. Labib K. Yu C. Zhang Z. The Mcm2-Ctf4-Polα axis facilitates parental histone H3-H4 transfer to lagging strands.Mol. Cell. 2018; 72 (30244834): 140-15110.1016/j.molcel.2018.09.001Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 7Huang H. Strømme C.B. Saredi G. Hödl M. Strandsby A. González-Aguilera C. Chen S. Groth A. Dinshaw. Patel D.J. A unique binding mode enables MCM2 to chaperone histones H3–H4 at replication forks.Nat. Struct. Mol. Biol. 2015; 22 (26167883): 618-62610.1038/nsmb.3055Crossref PubMed Scopus (109) Google Scholar, 8Clemente-Ruiz M. González-Prieto R. Félix P. Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks.PLoS Genet. 2011; 7 (22102830): e100237610.1371/journal.pgen.1002376Crossref PubMed Scopus (52) Google Scholar), thus ensuring efficient restoration of the proper epigenetic landscape. As lagging strand replication proceeds in the opposite direction of the replication fork, rebinding of transcription factors and reassembly of nucleosomes would form barriers to the lagging strand DNA polymerase δ (Pol δ). Indeed, nucleosomes appear to block Pol δ DNA synthesis both in vivo (9Smith D.J. Whitehouse I. Intrinsic coupling of lagging-strand synthesis to chromatin assembly.Nature. 2012; 483 (22419157): 434-43810.1038/nature10895Crossref PubMed Scopus (187) Google Scholar) and in vitro (10Devbhandari S. Jiang J. Kumar C. Whitehouse I. Remus D. Chromatin constrains the initiation and elongation of DNA replication.Mol. Cell. 2017; 65 (27989437): 131-14110.1016/j.molcel.2016.10.035Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and possibly limit strand displacement synthesis to prevent excessive re-replication, whereas still allowing primer removal. However, in yeast long 5'-flaps can arise during Okazaki fragment maturation and be extended by the 5'-3' DNA helicase Pif1, necessitating the endonuclease activity of the nuclease/helicase Dna2 to cleave the extended flap (11Rossi M.L. Pike J.E. Wang W. Burgers P.M.J. Campbell J.L. Bambara R.A. Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal.J. Biol. Chem. 2008; 283 (18689797): 27483-2749310.1074/jbc.M804550200Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 12Pike J.E. Burgers P.M.J. Campbell J.L. Bambara R.A. Pif1 helicase lengthens some Okazaki fragment flaps necessitating Dna2 nuclease/helicase action in the two-nuclease processing pathway.J. Biol. Chem. 2009; 284 (19605347): 25170-2518010.1074/jbc.M109.023325Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The flaps extended by Pif1 in vivo can reach lengths of hundreds (13Liu B. Hu J. Wang J. Kong D. Direct visualization of RNA-DNA primer removal from Okazaki fragments provides support for flap cleavage and exonucleolytic pathways in eukaryotic cells.J. Biol. Chem. 2017; 292 (28159842): 4777-478810.1074/jbc.M116.758599Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) to thousands (14Rossi S.E. Foiani M. Giannattasio M. Dna2 processes behind the fork long ssDNA flaps generated by Pif1 and replication-dependent strand displacement.Nat. Commun. 2018; 9: 1-1110.1038/s41467-018-07378-5Crossref PubMed Scopus (14) Google Scholar) of nucleotides, suggesting that during this process Pif1 may displace nucleosomes assembled on downstream Okazaki fragments. This possibility remains to be tested using reconstituted systems. Interestingly, genome wide analysis of Okazaki fragment junctions showed that their position correlates with the position of the binding sites of the general transcription factors Abf1, Reb1, and Rap1 (9Smith D.J. Whitehouse I. Intrinsic coupling of lagging-strand synthesis to chromatin assembly.Nature. 2012; 483 (22419157): 434-43810.1038/nature10895Crossref PubMed Scopus (187) Google Scholar), consistent with these being a barrier to the progression of lagging strand DNA synthesis. Indeed, we showed that in vitro a single Rap1 tightly bound to a high-affinity DNA-binding site is a strong block to the strand displacement DNA synthesis activity of yeast DNA polymerase (Pol) and DNA replication through a single or an array of DNA-bound Rap1 molecules requires the helicase activity of Pif1 (15Koc K.N. Singh S.P. Stodola J.L. Burgers P.M. Galletto R. Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase δ.Nucleic Acids Res. 2016; 44 (27001517): 3811-381910.1093/nar/gkw181Crossref PubMed Scopus (19) Google Scholar). It remains to be tested whether other transcription factors are also a strong block to DNA synthesis by Pol δ, and, if so, which ones impart a requirement for the activity of the Pif1 helicase for efficient DNA synthesis by Pol δ. Here we show that Saccharomyces cerevisiae Reb1 and Tbf1 bound to dsDNA are both strong polar blocks to the strand displacement DNA synthesis activity of Pol δ. However, binding of the ssDNA-binding protein RPA to a 5'-ssDNA flap of the displaced strand is sufficient to stimulate strand displacement DNA synthesis by Pol δ through the block imparted by a bound Tbf1. The same is not true for Reb1, which remains a strong block even in the presence of RPA and requires the activity of the Pif1 helicase for its removal. These findings suggest that a subset of DNA-bound transcription factors may necessitate the activity of the Pif1 helicase for efficient progression of replication. We also show that a positioned nucleosome is a strong block to both strand displacement DNA synthesis by Pol δ and FEN1-mediated nick translation, requiring both the ssDNA-binding protein RPA and Pif1 helicase for through-replication. In the presence of the FEN1 nuclease, Pif1 no longer stimulates DNA synthesis through a nucleosome, as FEN1 removes the entry point for the helicase. On the other hand, whereas RPA bound to a 5'-flap only moderately inhibited FEN1, it was sufficient to promote Pif1 unwinding through a protein block, even in the presence of FEN1. We propose that one function of FEN1 during Okazaki fragment maturation is to prevent the potentially deleterious activity of Pif1. In vitro primer extension assays were performed with DNA substrates that contain a 1-nt gap to monitor successful base incorporation by the polymerase, and a downstream duplex region with a centrally positioned sequence recognition motif to direct binding of each transcription factor tested. The substrates also contain a 25-nt long 5' poly(dT) flap that prevents PCNA from sliding off the substrate, whereas also providing a binding site for RPA and an entry point for Pif1 5'-3' helicase. The scheme for the assay is depicted in Fig. 1A. Briefly, PCNA is loaded on the DNA substrate by RFC, followed by the binding of the specific transcription factor being tested, in the presence or absence of the single-stranded DNA-binding protein RPA. DNA synthesis is initiated by the addition of Pol δ and dNTPs and the helicase when tested. The formation of the extension products from the labeled primer is monitored over time. WT Pol δ is stalled by Reb1 bound to the downstream duplex (Fig. S1A). Because of its exonuclease activity, the enzyme idles, cyclically cleaving and resynthesizing DNA close to the block, and results in a distribution of stalled products, which makes their analysis difficult. Thus, we used an exonuclease-deficient variant of Pol δ (Pol δDV) to better quantify stall sites and fractions of extension products. Fig. 1, B and C, show representative primer extension assays and the quantifications of the fraction of full-length DNA synthesis products, respectively. The presence of Reb1 bound to the downstream duplex inhibits formation of full-length extension products, with most of the DNA synthesis stalling 2-3 nt prior to the Reb1 recognition sequence motif (Fig. 1, B, central panel, and D, forward orientation). Thus, Reb1 is a strong block to the strand displacement DNA synthesis activity of Pol δDV. Importantly, whereas binding of RPA to the 5'-flap of the substrate stimulates DNA synthesis by Pol δ (Fig. S1B), this stimulation is not sufficient to allow replication through the block imparted by the DNA-bound Reb1 (Fig. 1, B and C). Furthermore, Reb1 bound in a reverse orientation relative to the directionality of DNA synthesis still significantly delays Pol δDV (Fig. 1E and Fig. S1C). However, when bound in the reverse orientation Reb1 is a weaker replication block compared with the forward orientation, as indicated by both the larger fraction of full-length DNA synthesis products (Fig. 1E) and by the transient accumulation of the intermediate stalling products (Fig. 1D). Independent of orientation, it is only with the addition of the 5'-3' DNA helicase Pif1 that efficient DNA replication occurs past the Reb1 block, consistent with Pif1 removing the DNA-bound Reb1 downstream of the advancing polymerase. The requirement of Pif1 for efficient DNA synthesis by Pol δ past the Reb1 block is similar to what we reported for Rap1 (15Koc K.N. Singh S.P. Stodola J.L. Burgers P.M. Galletto R. Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase δ.Nucleic Acids Res. 2016; 44 (27001517): 3811-381910.1093/nar/gkw181Crossref PubMed Scopus (19) Google Scholar), and is consistent with the activity of a 5'-3' helicase being required whenever DNA replication must proceed timely and efficiently through a strong protein barrier. However, this observation raises the question of whether any protein tightly bound to DNA is a block to DNA synthesis and, thus, requires the helicase activity of Pif1 for its removal. To address this question we tested whether this conclusion would also hold true for the general transcription factor Tbf1 that tightly binds to DNA (16Koering C.E. Fourel G. Binet-Brasselet E. Laroche T. Klein F. Gilson E. Identification of high affinity Tbf1p-binding sites within the budding yeast genome.Nucleic Acids Res. 2000; 28 (10871401): 2519-252610.1093/nar/28.13.2519Crossref PubMed Scopus (41) Google Scholar). Similar to what we observed for Reb1, the DNA-bound Tbf1 is also a block to the strand displacement DNA synthesis activity of Pol δDV, with one of the DNA-bound orientations displaying a higher ability to impede DNA synthesis (Fig. 2). However, in stark contrast to the Reb1 block, binding of RPA to the 5'-flap of the substrate and its stimulation of DNA synthesis is enough to allow replication past the Tbf1 block, to the same extent as Pif1 stimulated DNA synthesis (Fig. 2). These findings suggest that, in the presence of abundant RPA, not all proteins that are tightly bound to DNA would pose a significant barrier to DNA replication and require the activity of a helicase for their removal. Next, we tested whether a tightly positioned nucleosome would block DNA replication, and whether the helicase activity of Pif1 would be needed to remove or reposition the nucleosome to allow DNA synthesis to proceed. For this, we designed a DNA substrate containing the strong nucleosome positioning 601 Widom sequence (17Lowary P.T. Widom J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.J. Mol. Biol. 1998; 276 (9514715): 19-4210.1006/jmbi.1997.1494Crossref PubMed Scopus (1071) Google Scholar) placed 20 bp from a nick, followed by either a short (3 bp) or long (50 bp) tail to monitor potential sliding of the nucleosome toward the end of the substrate (Fig. 3A). As the DNA substrate is Cy3 labeled prior to nucleosome assembly, it is important that for our experiments all free DNA is assembled into nucleosomes to avoid the contribution from unimpeded DNA synthesis on bare DNA. As shown in Fig. S2A, nucleosome assembly was complete, as no free DNA was detected following nucleosome assembly. On these DNA substrates, Pol δWT showed limited strand displacement synthesis activity, even in the presence of RPA, and failed to reach the nucleosome (Fig. S2B). Therefore, exonuclease-deficient Pol δDV was again used to precisely quantify the polymerase stall sites. On a nucleosome-free substrate, Pol δDV carries out strand displacement DNA synthesis as indicated by formation of both intermediate and full-length DNA synthesis products (Fig. 3B, panel 1). Again, binding of RPA to the growing 5'-flap formed by Pol δDV stimulated strand displacement DNA synthesis, as indicated by the extension of all the intermediate products to full-length (Fig. 3B, panel 2, and Fig. S2C). However, on a nucleosome-bound substrate, few full-length products are formed, and Pol δDV is clearly halted at the front edge of the nucleosome (Fig. 3B, panel 3). Importantly, RPA does not stimulate DNA synthesis through the nucleosome, although it promotes synthesis about 8 nt further into it (Fig. 3B, panels 3 and 4). These data are presented quantitatively by monitoring the rate of accumulation of full-length DNA synthesis products (Fig. S2C) and by quantifying the band intensities of all the DNA synthesis products for each reaction condition at 30 min (Fig. 3C). Next, we tested whether the unwinding activity of Pif1 would be sufficient to remove the stably positioned nucleosome and allow through-replication. Surprisingly, Pif1 was able to disrupt the stably bound nucleosome and promote through-replication, especially in the presence of RPA (Fig. 3B, panels 5 and 6). In the absence of RPA, Pif1 stimulates about 10% formation of full-length product (Fig. S2C), and DNA synthesis by Pol δDV stalls just before the nucleosome dyad (Fig. 3B, panel 5). On the other hand, the combined unwinding activity of Pif1 and binding of RPA to the growing 5' ssDNA flap resulted in the near elimination of stalling just prior to the nucleosome dyad (Fig. 3C, panel 6), and in a rate of accumulation of full-length DNA synthesis product that is similar to the one observed for naked DNA (Fig. S2C). The stimulation of DNA synthesis through a nucleosome by Pif1 raises the question of whether the nucleosome is simply being removed as Pif1 unwinds the duplex DNA ahead of the polymerase or, because of the linear nature of the DNA substrate used, the nucleosome is being pushed off the end of the DNA. To test this latter possibility, we generated an otherwise identical DNA substrate that contains an extended region of 50 bp rather than 3 bp past the nucleosome positioning sequence. We reasoned that if the nucleosome was pushed downstream by Pif1 along the longer DNA, synthesis should stall further into the duplex, resulting in an offset of the position of the intermediate DNA synthesis products. In the absence of Pif1 and independent of RPA, Pol δDV stalled at the same positions on either substrate (Fig. S3A, panels 1-4). These data indicate that the nucleosome is properly positioned at the same location on both DNA substrates and that the polymerase itself cannot push the nucleosome. Independent of the downstream length past the 601 sequence, DNA synthesis by Pol δDV in the presence of Pif1 stalled at similar positions (Fig. S3A, panels 5-8). We take this as an indication that the nucleosome is not being pushed downstream by Pif1 but, rather, it is removed during unwinding. The mechanism by which this happens remains to be elucidated. The results in the previous sections suggest that the ability of the Pif1 helicase to efficiently disrupt tightly bound proteins and nucleosomes should allow unperturbed DNA replication through most protein barriers. However, this activity of Pif1 may need to be controlled to avoid potentially deleterious consequences during DNA replication, such as excessive flap generation and unwarranted removal of nucleosomes. Nucleosome disruption during lagging strand replication could lead to loss of epigenetic information or futile re-replication of downstream Okazaki fragments. During Okazaki fragment maturation, FEN1 travels along with Pol δ to promote nick translation: a repetitive cycle of short-range strand displacement by Pol δ and flap cleavage by FEN1, as depicted in Fig. 3A (18Stodola J. Burgers P. Resolving individual steps of Okazaki-fragment maturation at a millisecond timescale.Nat. Struct. Mol. Biol. 2016; 23 (27065195): 402-40810.1038/nsmb.3207Crossref PubMed Scopus (50) Google Scholar). Thus, we sought to test whether the activity of the FEN1 nuclease would be sufficient to remove the entry point for Pif1 and thereby prevent excessive replication through barriers during nick translation. To measure nick translation under physiologically relevant conditions, primer extension assays were performed with WT Pol δ. On naked DNA, FEN1 allows Pol δWT to efficiently synthesize full-length DNA products (Fig. 4, B, panel 1, and D). However, on a nucleosome-bound substrate, nick translation was blocked at the edge of the nucleosome (Fig. 4C, panel 1), thus preventing synthesis of full-length products (Fig. 4E). These findings are in agreement with previous results (10Devbhandari S. Jiang J. Kumar C. Whitehouse I. Remus D. Chromatin constrains the initiation and elongation of DNA replication.Mol. Cell. 2017; 65 (27989437): 131-14110.1016/j.molcel.2016.10.035Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), and show that nick translation is halted by a strongly positioned nucleosome. Next, we performed the same assays in the presence of the Pif1 helicase. Without FEN1 present, Pif1 promotes replication by Pol δWT through a nucleosome (Fig. 4E and Fig. S2B). However, the situation is quite different when FEN1 is present in the reaction. FEN1 prevents Pif1 from stimulating Pol δWT DNA synthesis even on a nucleosome-free substrate (Fig. 4, B and C, compare panels 1 and 2). Nick translation by FEN1 and Pol δWT is characterized by many intermediate products as Pol δWT cyclically polymerizes a few nucleotides and idles to allow FEN1 cleavage of the short displaced flap. This patterning of DNA products was unaffected by the presence of Pif1. Furthermore, Pif1 had no effect on the rate of full-length product formation in reactions that contained FEN1 (Fig. 4, D and E). We interpret these results as indicative of FEN1 preventing Pif1 from acting on the DNA substrate by continuously cleaving the 5'-flap that is used as an entry point by Pif1. Thus, when Pol δ synthesizes DNA from a nick, the catalytic activity of FEN1 is sufficient to prevent stimulation of DNA synthesis by Pif1. FEN1 effectively prevented Pif1 stimulation of DNA synthesis through a nucleosome. However, during Okazaki fragment processing, long flaps can form (14Rossi S.E. Foiani M. Giannattasio M. Dna2 processes behind the fork long ssDNA flaps generated by Pif1 and replication-dependent strand displacement.Nat. Commun. 2018; 9: 1-1110.1038/s41467-018-07378-5Crossref PubMed Scopus (14) Google Scholar) and become inhibitory to FEN1 cleavage if bound by RPA (11Rossi M.L. Pike J.E. Wang W. Burgers P.M.J. Campbell J.L. Bambara R.A. Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal.J. Biol. Chem. 2008; 283 (18689797): 27483-2749310.1074/jbc.M804550200Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Thus, we tested the ability of FEN1 to protect against Pif1 stimulation on a substrate with a long 5'-flap in the presence or absence of RPA. To test this, we used the DNA substrate that contains a Reb1-binding site in the downstream dsDNA (Fig. 5) and all the experiments were performed in the presence of Reb1 to slow full-length product formation. Interestingly, FEN1 has a slight stimulatory effect on DNA synthesis through Reb1, regardless of the absence (Fig. 5A) or presence (Fig. 5B) of RPA. This was unexpected because in the presence of RPA, FEN1 was expected to be inhibited and, thus, not be able to stimulate Pol δWT. To test whether RPA inhibits FEN1 in our system, the 5'-flap was radiolabeled and cleavage by FEN1 was monitored directly (Fig. S3B). FEN1 cleaves at the base of the 5'-flap; however, at the low RPA concentration used in our reactions RPA only partially inhibits FEN1. Although our findings may appear to be at odds with the commonly accepted idea in the field that RPA inhibits FEN1, the RPA concentration dependence of FEN1 inhibition showed that a large excess of RPA relative to the DNA is needed for significant inhibition to occur (19Henry R.A. Balakrishnan L. Tan Ying-Lin S. Campbell J.L. Bambara R.A. Components of the secondary pathway stimulate the primary pathway of eukaryotic Okazaki fragment processing.J. Biol. Chem. 2010; 285 (20628185): 28496-2850510.1074/jbc.M110.131870Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 20Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. On the roles of Saccharomyces cerevisiae Dna2p and Flap endonuclease 1 in Okazaki fragment processing.J. Biol. Chem. 2004; 279 (14747468): 15014-1502410.1074/jbc.M313216200Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Regardless, we found that RPA has a significant impact in promoting Pif1 unwinding over FEN1 cleavage. Without RPA, FEN1 and Pif1 compete for access to the 5'-flap and results in full-length product formation being intermediate of what achieved with Pif1 or FEN1 alone (Fig. 5A). However, in the presence of RPA, reactions containing Pif1 behave identically whether or not FEN1 is present (Fig. 5B), consistent with the finding that RPA inhibition of FEN1 was amplified by Pif1 (11). This result suggests that weak inhibition of FEN1 by RPA bound to a long flap is sufficient to favor Pif1 binding and unwinding, and suggests that for FEN1 to be effective in cleaving the 5' entry point for Pif1 it must do so before a long flap can form and interact with RPA. Together, these results reveal that the cyclical actions of Pol δWT and FEN1 during nick translation are sufficient to prevent Pif1 from acting on the DNA substrate. However, the presence of a long RPA bound 5'-flap blocks FEN1 from cleaving the 5'-flap and protecting the substrate from Pif1 entry. In this work, we showed that two transcription factors from S. cerevisiae, Reb1 and Tbf1, are strong blocks to DNA replication by Pol δ. Removal of the block imparted by Reb1 requires the activity of the Pif1 helicase to allow progression of DNA synthesis, similar to what we had observed for Rap1 (15Koc K.N. Singh S.P. Stodola J.L. Burgers P.M. Galletto R. Pif1 removes a Rap1-dependent barrier to the strand displacement activity of DNA polymerase δ.Nucleic Acids Res. 2016; 44 (27001517): 3811-381910.1093/nar/gkw181Crossref PubMed Scopus (19) Google Scholar). Unlike Rap1, which forms a closed complex on DNA (21König P. Giraldo R. Chapman L. Rhodes D. The crystal structure of the DNA-binding domain of yeast RAP1 in complex with telomeric DNA.Cell. 1996; 85 (8620531): 125-13610.1016/S0092-8674(00)81088-0Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), crystal structures of Schizosaccharomyces pombe Reb1 suggest that Reb1 binds DNA without forming a closed complex (22Jaiswal R. Choudhury M. Zaman S. Singh S. Santosh V. Bastia D. Escalante C.R. Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe.Proc. Natl. Acad. Sci. U.S.A. 2016; 113 (27035982): E2267-E227610.1073/pnas.1525465113Crossref PubMed Scopus (13) Google Scholar), yet we show here that it still forms a significant block to DNA synthesis that can only be removed by the activity of a helicase. On the other hand, whereas Tbf1 also forms a strong barrier to Pol δ DNA synthesis, the binding of RPA to the ssDNA flap of the substrate is all that is required to overcome the Tbf1 block and stimulate the DNA-synthesis activity of the polymerase. Thus, we conclude that in the absence of additional factors Rap1, Reb1, and Tbf1 are all strong barriers to the intrinsic strand displacement DNA-synthesis activity of Pol δ, independent of their DNA-binding affinity or specific configuration adopted on DNA. Interestingly, different additional factors are needed for their removal; either the simple binding of RPA and its stimulation of DNA synthesis or the ATP-dependent DNA-unwinding activity of an accessory helicase, such as Pif1. Although it remains to be determined how the DNA-binding affinity and/or specific DNA binding modes of transcription factors may contribute to defining their ability to act as a barrier to DNA replication, our data strongly suggest that not all transcription factors that can block Pol δ DNA synthesis will be a block to DNA replication when RPA is abundant. During DNA replication, nucleosomes are removed from in front of the replication fork and redeposited behind the fork, randomly distributed between the two daughter strands (3Groth A. Replicating chromatin: a tale of histones.Biochem. Cell Biol. 2009; 87 (19234523): 51-6310.1139/O08-102Crossref PubMed Scopus (20) Google Scholar, 4Annunziato A.T. The fork in the road: histone partitioning during DNA replication.Genes. 2015; 6 (26110314): 353-37110.3390/genes6020353Crossref PubMed Scopus (33) Google Scholar, 5Prado F. Maya D. Regulation of replication fork advance and stability by nucleosome assembly.Genes. 2017; 8: 4910.3390/genes8020049Crossref Scopus (19) Google Scholar, 6Gan H. Serra-Car
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