Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin

Tsurimoto, Toshiki; Melendy, Thomas; Stillman, Bruce

doi:10.1038/346534a0

Cited by 419 publications

(290 citation statements)

References 44 publications

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“…A role for pol ⑀ in replication late in S phase would be consistent with the observation that temperature-sensitive mutants of the cat- alytic subunit of pol ⑀ in S. cerevisiae, when shifted to the non-permissive temperature, slow chromosomal DNA synthesis gradually until they arrest with 2C of DNA (9). Although pol ⑀ is found associated with nascent cellular DNA (5) and is necessary for chromosomal DNA replication (8,10,14,65), it is not required for SV40 DNA replication (3,4) and cannot be immunoprecipitated with nascent SV40 DNA (5). pol ⑀ may be dispensable for the replication of viral DNA because it is akin to transcribed cellular DNA.…”

Section: Discussionsupporting

confidence: 59%

“…pol ␣ is clearly involved in the synthesis and extension of RNA primers during DNA replication initiation (reviewed in Ref. 1), whereas pol ␦ is responsible for elongation, as most clearly demonstrated in SV40 DNA replication (2,3). Although pol ⑀ is not required for SV40 replication (4,5), it does appear to have a role in chromosomal DNA replication (5)(6)(7)(8)(9)(10).…”

mentioning

confidence: 99%

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Human DNA Polymerase ε Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase

Fuss

Linn

2002

Journal of Biological Chemistry

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DNA polymerase ⑀ (pol ⑀) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol ⑀ was examined by indirect immunofluorescence, pol ⑀ appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol ⑀ foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol ⑀ foci were present throughout mitosis and the G 1 phase of cycling cells. It is hypothesized from these observations that pol ⑀ and PCNA have separate but associated functions early in S phase and that pol ⑀ participates with PCNA in DNA replication late in S phase.Although as many as 13 DNA polymerases are known to exist in eukaryotes, only 3 are thought to be involved in chromosomal replication; they are DNA polymerases (pol) 1 ␣, ␦, and ⑀. pol ␣ is clearly involved in the synthesis and extension of RNA primers during DNA replication initiation (reviewed in Ref. 1), whereas pol ␦ is responsible for elongation, as most clearly demonstrated in SV40 DNA replication (2, 3). Although pol ⑀ is not required for SV40 replication (4, 5), it does appear to have a role in chromosomal DNA replication (5-10).DNA pol ⑀ is well conserved in sequence and subunit composition among eukaryotes. Human pol ⑀ has four subunits, a large catalytic subunit, p261, and three associated subunits, p59, p12, and p17 (11,12). Likewise, Saccharomyces cerevisiae pol ⑀ has a catalytic subunit of 256 kDa, POL2, and has three associated subunits encoded by DPB2, DPB3, DPB4 (8, 13-15).Both catalytic subunits are divided into two domains linked by a protease-sensitive region, an N-terminal domain containing polymerase and exonuclease motifs and a 120-kDa C-terminal domain. The S. cerevisiae and human catalytic subunits are 39% identical overall and have 63% identity in the N-terminal domain (16). p59 and DPB2p have 26% overall identity (17), whereas p17 and p12 have 36.5 and 34% identity to Dpb4p and Dpb3p, respectively (12). Given the conservation of pol ⑀ sequence and subunit structure between yeast and humans, it is likely that their functions are also largely conserved.A role for pol ⑀ in chromosomal DNA replication has been established in higher eukaryotes and in yeast. The elongation step of DNA replication is impaired in Xenopus laevis egg extracts immunodepleted of pol ⑀ (6). Additionally, using a polymerase trap technique that tags a polymerase with its DNA product, Zlotkin et al. (5) find that pol ⑀ from monkey kidney cells associates with nascent chromosomal DNA but not nascent SV40 DNA. Likewise, a neutralizing antibody against human pol ⑀ inhibits chromosomal replication when microinjected into human fibroblasts but fails to inhibit SV40 DNA replication in vitro (7). In S. cerevisiae, disruptions of POL2 are lethal and result in the terminal dumbbell morphology characteristic of a defect in DNA replication (8). When shifted to the no...

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Section: Discussionsupporting

confidence: 59%

mentioning

confidence: 99%

Human DNA Polymerase ε Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase

Fuss

Linn

2002

Journal of Biological Chemistry

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“…This was an important point to consider, as another explanation for the resistance of the full NER reaction to inhibition by p21 could be that di erent DNA polymerases are normally used for NER and for replication, with one polymerase particularly resistant to inhibition by p21. There is good evidence that replication of SV40 viral DNA in vitro requires pol d (Tsurimoto et al, 1990;Weinberg et al, 1990;Hurwitz et al, 1990), and that mammalian chromosomal replication requires both pol d and pol e (Zlotkin et al, 1996). It would be possible, for example, that NER normally functions with pol e and this enzyme is especially refractory to p21 inhibition.…”

Section: Resistance Of Ner In Human Cell Extracts To Inhibition By P21mentioning

confidence: 99%

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

et al. 1998

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The p21 Cdn1 protein (cip1/waf1/sdi1) plays an important role as an inhibitor of mammalian cell proliferation in response to DNA damage. By interacting with and inhibiting the function of cyclin-Cdk complexes, p21 can block entry into S phase. p21 can also directly inhibit replicative DNA synthesis by binding to the DNA polymerase sliding clamp factor PCNA. When cells are damaged and p21 is induced, DNA nucleotide excision repair (NER) continues, even though this pathway is PCNA-dependent. We investigated features of p21-resistant NER using human cell extracts. A direct endlabelling approach was used to measure the excision of damaged oligonucleotides by NER and no inhibition by p21 was found. By contrast, ®lling of the *30 nt gaps created by NER could be inhibited by pre-binding p21 to PCNA, but only when gap ®lling was uncoupled from incision. Binding p21 to PCNA could also inhibit ®lling of model 30 nt gaps by both puri®ed DNA polymerases d and e. When p21 was incubated in a cell extract before addition of PCNA, inhibition of repair synthesis was gradually relieved with time. This incubation gives p21 the opportunity to associate with other targets. As p21 blocks association of DNA polymerases with PCNA but does not prevent loading of PCNA onto DNA, repair gap ®lling can occur rapidly as soon as p21 dissociates from PCNA. A synthetic PCNA-binding p21 peptide was an e cient inhibitor of NER synthesis in cell extracts.

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“…Pol α-primase contains both priming and DNA polymerase activities, and left unchecked, the polymerase activity of Pol α-primase has been demonstrated to function with CMG to replicate both the leading and lagging strands (9,10). Pol α-primase was earlier shown to function with the SV40 T-antigen helicase to synthesizes both the leading and lagging strands of the SV40 genome (35). Limiting Pol α-primase function is important because the enzyme lacks a proofreading exonuclease, and thus has lower fidelity than either Pol e or Pol δ (reviewed in ref.…”

Section: Quality Control Of Polmentioning

confidence: 99%

Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

Schauer

O'Donnell

2017

Proc. Natl. Acad. Sci. U.S.A.

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The eukaryotic genome is primarily replicated by two DNA polymerases, Pol e and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol e and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol e on the lagging strand, and CMG protects Pol e against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ-PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol e binds CMG with a K d value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ-PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ-CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand.D uplication of genetic material is performed by a dynamic interplay of numerous different proteins, collectively referred to as the replisome, that orchestrate their actions to accomplish efficient, high-fidelity replication of both the leading and lagging strands (1-3). Eukaryotes possess several replisome factors that have no homologs in bacteria, and also require two different DNA polymerases for bulk leading and lagging strand synthesis, and Escherichia coli and its phages use identical copies of a DNA polymerase for both strands of the duplex (4). Thus far, the evolutionary purpose behind the additional complexity in eukaryotes is unclear.At the heart of the eukaryotic replisome is an 11-subunit helicase complex referred to as CMG (Cdc45-Mcm2-7-GINS) (5-7). Eukaryotes use three different multisubunit B-family DNA polymerases (Pol) for replication. Pol e and Pol δ copy the bulk of the genome, and Pol α-primase generates hybrid RNA-DNA primers of about 25 nucleotides and, unlike Pol e and Pol δ, it lacks 3′-5′ exonuclease (proofreading) activity. The replication factor C (RFC) clamp loader is used to load PCNA (proliferating cell nuclear antigen) clamps onto primed sites that endow the replicative polymerases with high processivity (8). The replication protein A (RPA) heterotrimer binds and protects the single-strand (ss) DNA of the lagging strand against nucleases, and helps melt secondary structure in ssDNA. Reconstitution of the core eukaryotic replisome using all thre...

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Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin

Cited by 419 publications

References 44 publications

Human DNA Polymerase ε Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase

Human DNA Polymerase ε Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase

Resistance of human nucleotide excision repair synthesis in vitro to p21Cdn1

Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

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