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Segregation of Replicative DNA Polymerases during S Phase

DNA POLYMERASE ϵ, BUT NOT DNA POLYMERASES α/δ, ARE ASSOCIATED WITH LAMINS THROUGHOUT S PHASE IN HUMAN CELLS*
Open AccessPublished:August 10, 2012DOI:https://doi.org/10.1074/jbc.M112.357996
      DNA polymerases (Pol) α, δ, and ϵ replicate the bulk of chromosomal DNA in eukaryotic cells, Pol ϵ being the main leading strand and Pol δ the lagging strand DNA polymerase. By applying chromatin immunoprecipitation (ChIP) and quantitative PCR we found that at G1/S arrest, all three DNA polymerases were enriched with DNA containing the early firing lamin B2 origin of replication and, 2 h after release from the block, with DNA containing the origin at the upstream promoter region of the MCM4 gene. Pol α, δ, and ϵ were released from these origins upon firing. All three DNA polymerases, Mcm3 and Cdc45, but not Orc2, still formed complexes in late S phase. Reciprocal ChIP of the three DNA polymerases revealed that at G1/S arrest and early in S phase, Pol α, δ, and ϵ were associated with the same nucleoprotein complexes, whereas in late S phase Pol ϵ and Pol α/δ were largely associated with distinct complexes. At G1/S arrest, the replicative DNA polymerases were associated with lamins, but in late S phase only Pol ϵ, not Pol α/δ, remained associated with lamins. Consistently, Pol ϵ, but not Pol δ, was found in nuclear matrix fraction throughout the cell cycle. Therefore, Pol ϵ and Pol α/δ seem to pursue their functions at least in part independently in late S phase, either by physical uncoupling of lagging strand maturation from the fork progression, or by recruitment of Pol δ, but not Pol ϵ, to post-replicative processes such as translesion synthesis or post-replicative repair.

      Introduction

      Three DNA polymerases (Pol)
      The abbreviations used are: Pol
      polymerase(s)
      PCNA
      proliferating cell nuclear antigen
      ORC
      origin recognition complex
      qPCR
      quantitative PCR
      UPR
      upstream promoter region.
      α, δ, and ϵ replicate the bulk of the eukaryotic genome (for reviews, see Refs.
      • Stillman B.
      DNA polymerases at the replication fork in eukaryotes.
      ,
      • Nick McElhinny S.A.
      • Gordenin D.A.
      • Stith C.M.
      • Burgers P.M.
      • Kunkel T.A.
      Division of labor at the eukaryotic replication fork.
      ,
      • Johansson E.
      • Macneill S.A.
      The eukaryotic replicative DNA polymerases take shape.
      ). Pol α is unique among DNA polymerases by having an intrinsic primase. It is therefore able to start DNA synthesis de novo (reviewed in Refs.
      • Nasheuer H.P.
      • Grosse F.
      DNA polymerase α-primase from calf thymus. Determination of the polypeptide responsible for primase activity.
      and
      • Nasheuer H.P.
      • Smith R.
      • Bauerschmidt C.
      • Grosse F.
      • Weisshart K.
      Initiation of eukaryotic DNA replication. Regulation and mechanisms.
      ). The primase acts as a DNA-dependent RNA polymerase synthesizing an RNA primer of about 10 bases long, which is then extended by the DNA polymerase activity of Pol α complex to about 30 bases. For duplication of simian virus 40 (SV40) DNA, a classic model system for eukaryotic DNA replication, replication factor C is specifically bound to these primers and expels Pol α. Replication factor C then loads the ring-shaped proliferating cell nuclear antigen (PCNA) to form a sliding clamp around the double-stranded DNA at the primer end, and recruits Pol δ, which synthesizes both leading strand DNA and Okazaki fragments of the lagging strand, the latter being then processed to a continuous strand (for review, see Ref.
      • Waga S.
      • Stillman B.
      The DNA replication fork in eukaryotic cells.
      ). Besides Pol α and δ, a third large DNA polymerase, Pol ϵ, was found to be essential for yeast Sacharomyces cerevisiae (
      • Morrison A.
      • Araki H.
      • Clark A.B.
      • Hamatake R.K.
      • Sugino A.
      A third essential DNA polymerase in S. cerevisiae.
      ), and it was found to be involved in synthesis of chromosomal DNA in human cells (
      • Zlotkin T.
      • Kaufmann G.
      • Jiang Y.
      • Lee M.Y.
      • Uitto L.
      • Syväoja J.
      • Dornreiter I.
      • Fanning E.
      • Nethanel T.
      DNA polymerase ϵ may be dispensable for SV40, but not cellular, DNA replication.
      ,
      • Pospiech H.
      • Kursula I.
      • Abdel-Aziz W.
      • Malkas L.
      • Uitto L.
      • Kastelli M.
      • Vihinen-Ranta M.
      • Eskelinen S.
      • Syväoja J.E.
      A neutralizing antibody against human DNA polymerase ϵ inhibits cellular but not SV40 DNA replication.
      ,
      • Bermudez V.P.
      • Farina A.
      • Raghavan V.
      • Tappin I.
      • Hurwitz J.
      Studies on human DNA polymerase ϵ and GINS complex and their role in DNA replication.
      ). It is also required for efficient DNA synthesis in Xenopus egg extracts (
      • Shikata K.
      • Sasa-Masuda T.
      • Okuno Y.
      • Waga S.
      • Sugino A.
      The DNA polymerase activity of Pol ϵ holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts.
      ). It has been recently found that S. cerevisiae Pol δ and ϵ harboring mutations that confer specific mutation patterns to the enzymes, sign their mutational signatures to lagging and leading strand, respectively (
      • Nick McElhinny S.A.
      • Gordenin D.A.
      • Stith C.M.
      • Burgers P.M.
      • Kunkel T.A.
      Division of labor at the eukaryotic replication fork.
      ,
      • Pursell Z.F.
      • Isoz I.
      • Lundström E.B.
      • Johansson E.
      • Kunkel T.A.
      Yeast DNA polymerase ϵ participates in leading-strand DNA replication.
      ,
      • Larrea A.A.
      • Lujan S.A.
      • Nick McElhinny S.A.
      • Mieczkowski P.A.
      • Resnick M.A.
      • Gordenin D.A.
      • Kunkel T.A.
      Genome-wide model for the normal eukaryotic DNA replication fork.
      ). Based on this evidence and on former work (for review, see Ref.
      • Pavlov Y.I.
      • Shcherbakova P.V.
      DNA polymerases at the eukaryotic fork. 20 Years Later.
      ) it is safe to conclude that Pol δ is a main player in synthesis of lagging strand DNA, whereas Pol ϵ is predominantly involved in the synthesis of the leading strand DNA.
      However, there is also evidence according to which the division of labor between Pol δ and ϵ may be more complex than a simple splitting between lagging and leading strands, respectively. The deletion of the domain containing polymerase and proofreading exonuclease motifs from S. cerevisiae causes growth and replication defects but the deletion is not lethal (
      • Kesti T.
      • Flick K.
      • Keränen S.
      • Syväoja J.E.
      • Wittenberg C.
      DNA polymerase ϵ catalytic domains are dispensable for DNA replication, DNA repair, and cell viability.
      ,
      • Dua R.
      • Levy D.L.
      • Campbell J.L.
      Analysis of the essential functions of the C-terminal protein/protein interaction domain of Saccharomyces cerevisiae Pol ϵ and its unexpected ability to support growth in the absence of the DNA polymerase domain.
      ), indicating that in this case, like in SV40 DNA replication, Pol δ is able to synthesize both strands. Furthermore, when the proofreading activity of Pol δ is mutationally inactivated, the mutation rate is significantly higher than in cells having analogous mutation in Pol ϵ (
      • Morrison A.
      • Johnson A.L.
      • Johnston L.H.
      • Sugino A.
      Pathway correcting DNA replication errors in Saccharomyces cerevisiae.
      ,
      • Morrison A.
      • Sugino A.
      The 3′–>5′ exonucleases of both DNA polymerases delta and epsilon participate in correcting errors of DNA replication in Saccharomyces cerevisiae.
      ). Amino acid substitutions in the polymerase domain of Pol δ also seem to generate a higher increase in the mutation rates and cause more severe growth defects than analogous amino acid substitutions in Pol ϵ (
      • Pavlov Y.I.
      • Shcherbakova P.V.
      • Kunkel T.A.
      In vivo consequences of putative active site mutations in yeast DNA polymerases α, ϵ, δ, and ζ.
      ). Further evidence conflicting with the current model comes from studies of human cells. We previously found that (i) a neutralizing antibody against Pol ϵ inhibits DNA synthesis in permeabilized nuclei more efficiently in the early S phase than in the late S phase, whereas the contrary is true for antibodies against Pol δ, and that (ii) trapping of Pol ϵ to nascent DNA remained nearly constant throughout the S phase, whereas Pol δ was three to four times more intensely cross-linked to nascent DNA in late compared with early S phase, and that (iii) the chromatin-bound fraction of Pol δ, unlike Pol ϵ, increased in the late S phase (
      • Rytkönen A.K.
      • Vaara M.
      • Nethanel T.
      • Kaufmann G.
      • Sormunen R.
      • Läärä E.
      • Nasheuer H.P.
      • Rahmeh A.
      • Lee M.Y.
      • Syväoja J.E.
      • Pospiech H.
      Distinctive activities of DNA polymerases during human DNA replication.
      ). These results suggest that the contribution of Pol δ to DNA synthesis increases toward the late S phase, whereas that of Pol ϵ either decreases or remains constant. In contrast, Fuss and Linn (
      • Fuss J.
      • Linn S.
      Human DNA polymerase ϵ colocalizes with proliferating cell nuclear antigen and DNA replication late, but not early, in S phase.
      ) proposed that Pol ϵ acts in the replication of heterochromatin during late S phase based on the observation that in immunofluorescense microscopy, the enzyme is neighboring PCNA foci and sites of DNA synthesis in early S phase but co-localizes with these sites in late S phase. Our previous study also suggested that ultrastructural localization of the Pol δ and ϵ were essentially distinct although minor colocalization was also detected (
      • Rytkönen A.K.
      • Vaara M.
      • Nethanel T.
      • Kaufmann G.
      • Sormunen R.
      • Läärä E.
      • Nasheuer H.P.
      • Rahmeh A.
      • Lee M.Y.
      • Syväoja J.E.
      • Pospiech H.
      Distinctive activities of DNA polymerases during human DNA replication.
      ). Depletion of the activity of Pol δ or ϵ in higher eukaryotes causes distinct defects for genome duplication (
      • Bermudez V.P.
      • Farina A.
      • Raghavan V.
      • Tappin I.
      • Hurwitz J.
      Studies on human DNA polymerase ϵ and GINS complex and their role in DNA replication.
      ,
      • Fukui T.
      • Yamauchi K.
      • Muroya T.
      • Akiyama M.
      • Maki H.
      • Sugino A.
      • Waga S.
      Distinct roles of DNA polymerases δ and ϵ at the replication fork in Xenopus egg extracts.
      ), arguing for different contributions to DNA replication. All these observations were not expected if Pol ϵ and δ are part of the same replication fork complex, acting on the leading and the lagging strands, respectively, in a similar manner as Pol III in Escherichia coli cells (for review, see Ref.
      • McHenry C.S.
      DNA replicases from a bacterial perspective.
      ). They could still be explained if (i) Pol δ is involved in delayed maturation of accumulating Okazaki fragments or both strands independent of Pol ϵ, (ii) Pol δ, but not Pol ϵ is increasingly involved in post-replicative processes such as DNA translesion synthesis or post-replicative DNA recombination, or if other than in yeast, (iii) the share of labor of Pol δ and ϵ has changed from yeast to human, i.e. human Pol ϵ acts more at early origins of replication and Pol δ more at late origins of replication on both strands. To address these questions we applied chromatin immunoprecipitation (ChIP) techniques and quantitative PCR to study association and release of the three replicative DNA polymerases, Pol α, δ, and ϵ, with DNA from two origins of replication, the lamin B2 (LB2) gene origin and the upstream promoter region (UPR) of MCM4 gene, firing at 0 and 2 h, respectively, after S phase entry, and to study co-existence of the three replicative DNA polymerases, origin recognition complex (ORC) subunit Orc2, a component of the Mcm-helicase complex Mcm3, and cell division control protein Cdc45 in cross-linked nucleoprotein complexes during S phase. We also studied the presence of lamins A/C in these complexes. The results reveal that Pol α, δ, and ϵ are loaded to and released from both origins of replication during S phase. In G1/S arrested cells Pol α, δ, and ϵ are present in highly purified nucleoprotein complexes containing 200–1000-base pair long DNA fragments and they are associated with lamins. In late S phase Pol α and δ are segregated from Pol ϵ and lamins A/C, whereas Pol ϵ remains associated with lamins. Based on this study and other studies that have been previously published we propose a model according to which Pol ϵ bound to nuclear matrix synthesizes the leading strand and Pol δ synthesizes the lagging strand, but the latter partly trails behind to process still immature lagging strand DNA, and possibly also leading strand DNA, or fulfills post-replicative tasks such as DNA translesion synthesis after Pol ϵ has essentially completed its job.

      DISCUSSION

      Applying the ChIP technique, we found that in HeLa cells all three replicative DNA polymerases, Pol α, δ, and ϵ, are bound to and released from both early firing LB2 origin and MCM4 UPR origin that was found to fire 2 h later in mid-S phase (FIGURE 1, FIGURE 2). Nucleoprotein complexes purified by immunoprecipitation with Pol α, δ, or ϵ antibodies from S phase cells were highly enriched with DNA fragments representing the two origins at the time preceding the firing, followed by a rapid decrease after firing. Therefore, both replicases are loaded to the two origins and disappear together with Mcm3, when replication forks have moved away from the origin. This is consistent with studies in yeast suggesting that Pol ϵ acts mainly at leading strand and Pol δ mainly at lagging strand synthesis (for review, see Ref.
      • Johansson E.
      • Macneill S.A.
      The eukaryotic replicative DNA polymerases take shape.
      ).
      All three DNA polymerases were associated with the same nucleoprotein complexes in G1/S-arrested cells, as can be expected if they all bind to origins of replication and are associated with each other or are even physically coupled at the replication fork at this time. In late S phase the three polymerases are all still likely to be at replication forks, as shown by the presence of Mcm3 and Cdc45 proteins in nucleoprotein complexes purified by immunoprecipitation with cognate Pol antibodies (Fig. 3A). However, in late S phase Pol δ and α seem to be essentially devoid of Pol ϵ, and vice versa, suggesting that Pol α/δ and Pol ϵ are now mainly associated with distinct complexes. This suggests that Pol δ and ϵ are physically uncoupled in late S phase. This is consistent with the fact that Pol ϵ alone remains associated with nuclear matrix, obviously through lamin A/C, whereas Pol α and δ are only associated with lamins in early S phase, most likely as components of replication complexes.
      How can the lack of association between Pol α/δ and Pol ϵ in late S phase be explained? First, the share of labor could be different in late compared with early S phase, such that late S phase forks have a different DNA polymerase composition. The co-localization of Pol ϵ with sites of DNA synthesis in late, but not early S phase (
      • Fuss J.
      • Linn S.
      Human DNA polymerase ϵ colocalizes with proliferating cell nuclear antigen and DNA replication late, but not early, in S phase.
      ), would be consistent with an augmented role of Pol ϵ in late S phase replication. Nevertheless, profiling of replication errors generated by an asymmetric mutator variant of Pol δ in the budding yeast indicate that Pol δ synthesizes the lagging-strand throughout the genome (
      • Larrea A.A.
      • Lujan S.A.
      • Nick McElhinny S.A.
      • Mieczkowski P.A.
      • Resnick M.A.
      • Gordenin D.A.
      • Kunkel T.A.
      Genome-wide model for the normal eukaryotic DNA replication fork.
      ), and argues strongly against such a model. We found in a previous study that in late S phase, Pol δ is associated more strongly with newly synthesized DNA and chromatin than Pol ϵ, and that replication in isolated nuclei is more strongly inhibited in late S than in early S phase by a monoclonal antibody inhibiting Pol δ (
      • Rytkönen A.K.
      • Vaara M.
      • Nethanel T.
      • Kaufmann G.
      • Sormunen R.
      • Läärä E.
      • Nasheuer H.P.
      • Rahmeh A.
      • Lee M.Y.
      • Syväoja J.E.
      • Pospiech H.
      Distinctive activities of DNA polymerases during human DNA replication.
      ). Therefore, alternatively, this can be explained in a manner proposed by Pavlov and Shcherbakova (
      • Pavlov Y.I.
      • Shcherbakova P.V.
      DNA polymerases at the eukaryotic fork. 20 Years Later.
      ). In their model Pol ϵ in principle replicates the leading strand, but is switched to Pol δ at pause sites or sites of translesion synthesis. In this way, Pol ϵ would synthesize most of the leading strand, but Pol δ would synthesize the parts of the leading strand after replication fork arrest and the restart of DNA synthesis. The fact that the Pol domain of Pol ϵ is not essential for viability of yeast (
      • Kesti T.
      • Flick K.
      • Keränen S.
      • Syväoja J.E.
      • Wittenberg C.
      DNA polymerase ϵ catalytic domains are dispensable for DNA replication, DNA repair, and cell viability.
      ) and that Pol ϵ is not needed for SV40 DNA replication (
      • Waga S.
      • Stillman B.
      The DNA replication fork in eukaryotic cells.
      ,
      • Zlotkin T.
      • Kaufmann G.
      • Jiang Y.
      • Lee M.Y.
      • Uitto L.
      • Syväoja J.
      • Dornreiter I.
      • Fanning E.
      • Nethanel T.
      DNA polymerase ϵ may be dispensable for SV40, but not cellular, DNA replication.
      ,
      • Pospiech H.
      • Kursula I.
      • Abdel-Aziz W.
      • Malkas L.
      • Uitto L.
      • Kastelli M.
      • Vihinen-Ranta M.
      • Eskelinen S.
      • Syväoja J.E.
      A neutralizing antibody against human DNA polymerase ϵ inhibits cellular but not SV40 DNA replication.
      ) are two examples of the ability of Pol δ to substitute for Pol ϵ at the leading strand. Alternatively, it is possible that replicative Pol α, δ, and ϵ in late S phase are increasingly involved in tasks that are no longer closely associated with the replication fork. It has been reported that a considerable amount of DNA translesion synthesis may be performed after and independent of DNA replication (
      • Karras G.I.
      • Jentsch S.
      The RAD6 DNA damage tolerance pathway operates uncoupled from the replication fork and is functional beyond S phase.
      ). There may also be a differential requirement of DNA polymerases, e.g. for the termination of DNA replication, or for the rescue of stalled and reversed forks by recombinational processes.
      Finally, Pol δ and ϵ may be physically separated in late S phase but are still synthesizing DNA at the lagging and leading strand of the same forks. This view is supported by the fact that all three replicative polymerases are associated with Mcm3 in the ChIP of highly purified nucleoprotein fractions (Fig. 3). Furthermore, β-satellite repeats become enriched in ChIP of Mcm3 as well as Pol α, δ, and ϵ 4 h after release from the thymidine block, consistent with the β-satellites being replicated late in S phase (
      • Ten Hagen K.G.
      • Cohen S.N.
      Timing of replication of β satellite repeats of human chromosomes.
      ) under participation of all three replicative polymerases. We consider the possibility that synthesis of the lagging strand is distributive, i.e. a new molecule of Pol α and Pol δ is recruited from a nucleoplasmic pool, and that the lagging strand replication machinery may be released from the fork during initiation of the following lagging strand. In this way, maturation of several successive Okazaki fragments may be ongoing simultaneously, and Pol δ molecules may remain associated with chromatin after completion and joining of Okazaki fragments. Such a model would explain both the increasing association of Pol δ with chromatin (Fig. 5B) as well as the increased involvement in DNA synthesis as S phase progresses (
      • Rytkönen A.K.
      • Vaara M.
      • Nethanel T.
      • Kaufmann G.
      • Sormunen R.
      • Läärä E.
      • Nasheuer H.P.
      • Rahmeh A.
      • Lee M.Y.
      • Syväoja J.E.
      • Pospiech H.
      Distinctive activities of DNA polymerases during human DNA replication.
      ). In contrast, the leading strand would be synthesized largely progressively by the same Pol ϵ molecule attached to the nuclear scaffold. There is no evidence on heterodimer formation by Pol δ and ϵ (see e.g. Ref.
      • Rytkönen A.K.
      • Hillukkala T.
      • Vaara M.
      • Sokka M.
      • Jokela M.
      • Sormunen R.
      • Nasheuer H.P.
      • Nethanel T.
      • Kaufmann G.
      • Pospiech H.
      • Syväoja J.E.
      DNA polymerase ϵ associates with the elongating form of RNA polymerase II and nascent transcripts.
      ) that would represent a human counterpart of the E. coli Pol III holoenzyme dimer or trimer that is stably associated with the core replication proteins (
      • McInerney P.
      • Johnson A.
      • Katz F.
      • O'Donnell M.
      Characterization of a triple DNA polymerase replisome.
      ,
      • Georgescu R.E.
      • Yao N.Y.
      • O'Donnell M.
      Single-molecule analysis of the Escherichia coli replisome and use of clamps to bypass replication barriers.
      ) and carries out simultaneous synthesis of both strands. Pol α, δ, and ϵ are all accumulated at replication roadblocks (
      • Pacek M.
      • Tutter A.V.
      • Kubota Y.
      • Takisawa H.
      • Walter J.C.
      Localization of MCM2–7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication.
      ), but they did not form a stable complex with a replication progression complex (
      • Gambus A.
      • Jones R.C.
      • Sanchez-Diaz A.
      • Kanemaki M.
      • van Deursen F.
      • Edmondson R.D.
      • Labib K.
      GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks.
      ). And although coupling of Pol α and ϵ with the Cdc45-Mcm2–7-GINS (CMG) helicase complex has been described (
      • Lou H.
      • Komata M.
      • Katou Y.
      • Guan Z.
      • Reis C.C.
      • Budd M.
      • Shirahige K.
      • Campbell J.L.
      Mrc1 and DNA polymerase ϵ function together in linking DNA replication and the S phase checkpoint.
      ,
      • Gambus A.
      • van Deursen F.
      • Polychronopoulos D.
      • Foltman M.
      • Jones R.C.
      • Edmondson R.D.
      • Calzada A.
      • Labib K.
      A key role for Ctf4 in coupling the MCM2–7 helicase to DNA polymerase α within the eukaryotic replisome.
      ), replicases are easily uncoupled from DNA unwinding after inhibition of DNA synthesis (
      • Pacek M.
      • Walter J.C.
      A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication.
      ) or after DNA damage (
      • Byun T.S.
      • Pacek M.
      • Yee M.C.
      • Walter J.C.
      • Cimprich K.A.
      Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.
      ). Therefore, transient association and dissociation of the replicative DNA polymerases with the CMG complex is likely to allow their flexible utilization for the duplication of complex human genome.
      It is possible that a combination of all three, different involvement of the Pol δ and ϵ in DNA replication-associated processes such as DNA translesion synthesis, increasing involvement of the replicative DNA polymerases in late S phase in tasks that are no longer closely associated with replication forks, and repeated recruitment of new Pol δ molecules from a nucleoplasmic pool for the lagging strand synthesis, account for the dynamics of Pols α, δ, and ϵ in their mutual association described here.

      Acknowledgments

      We are indebted to Leena Pääkkönen for excellent technical assistance.

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