Yeast DNA Polymerase ϵ Catalytic Core and Holoenzyme Have Comparable Catalytic Rates

Ganai, Rais Ahmad; Osterman, Pia; Johansson, Erik

doi:10.1074/jbc.m114.615278

Cited by 18 publications

(23 citation statements)

References 61 publications

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“…Interestingly, excision by p261N was enhanced by over 30-fold in the presence of a single mismatch, while hPol ε only experienced a 10-fold stimulation. Notably, a similar pattern was observed for enhancement of the excision rate constant of the yeast Pol ε catalytic subunit and holoenzyme [51]. This result provides evidence that the excision rate constant is mostly limited by the transfer of the DNA substrate from the polymerase active site to the exonuclease active site, as the excision rate constant of ssDNA at the exonuclease site should be independent of DNA duplex stability.…”

Section: Resultssupporting

confidence: 74%

“…2). Similarly, the rate constants for nucleotide incorporation by the yeast Pol ε catalytic subunit and holoenzyme were found to be nearly identical to each other [51]. Following the burst phase, hPol ε and p261N catalyzed additional product formation characterized by slower linear phases with rate constants of 0.047 ± 0.006 s −1 and 0.018 ± 0.004 s −1 , respectively.…”

Section: Resultsmentioning

confidence: 97%

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Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ

et al. 2015

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Numerous genetic studies have provided compelling evidence to establish DNA polymerase ε (Polε) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polε is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′ → 5′ exonuclease domain common to many replicative polymerases. In addition, Polε possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polε heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polε in vitro. However, similar studies of the human Polε heterote-tramer (hPolε) have been limited by the difficulty of obtaining hPolε in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolε from insect host cells has allowed for isolation of greater amounts of active hPolε, thus enabling a more detailed kinetic comparison between hPolε and an active N-terminal fragment of the hPolε catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolε. We observe that the small subunits increase DNA binding by hPolε relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolε is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolε and sway hPolε toward DNA synthesis rather than proofreading.

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Section: Resultssupporting

confidence: 74%

Section: Resultsmentioning

confidence: 97%

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ

et al. 2015

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“…(37) Recent pre-steady-state experiments have reported k pol values of ~250–300 s −1 with yeast and human pol epsilon, in line with the values reported for other B-family pols. (33, 38) However, the reaction buffer used in the pre-steady-state experiments with pol epsilon did not have any salt in them. Thus, the absolute values of the pre-steady-state kinetic constants reported might not represent what occurs at more physiological salt concentrations, such as the concentrations we have employed in our steady-state assays.…”

Section: Resultsmentioning

confidence: 99%

Evidence for the Kinetic Partitioning of Polymerase Activity on G-Quadruplex DNA

et al. 2015

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We have investigated the action of the human DNA polymerase epsilon (hpol ε) and eta (hpol η) catalytic cores on G-quadruplex (G4) DNA substrates derived from the promoter of the c-MYC proto-oncogene. The translesion enzyme hpol η exhibits a 6.2-fold preference for binding to G4 DNA relative to non-G4 DNA, while hpol ε binds both G4 and non-G4 substrates with near equal affinity. Kinetic analysis of single-nucleotide insertion by hpol η reveals that it is able to maintain greater than 25% activity on G4 substrates compared to non-G4 DNA substrates, even when the primer template junction is positioned directly adjacent to G22 (the first tetrad-associated guanine in the c-MYC G4 motif). Surprisingly, hpol η fidelity increases ~15-fold when copying G22. By way of comparison, hpol ε retains ~4% activity and has a 33-fold decrease in fidelity when copying G22. The fidelity of hpol η is ~100-fold more accurate than hpol ε when comparing the mis-insertion frequencies of the two enzymes opposite a tetrad-associated guanine. The kinetic differences observed for the B- and Y-family pols on G4 DNA support a model where a simple kinetic switch between replicative and TLS pols could help govern fork progress during G4 DNA replication.

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“…The replication clamp PCNA enhances not only the processivity of Pol δ but also its actual rate of catalysis, such that at saturating dNTP concentrations, Pol δ replicates at a rate of ~250 nt/sec (114). Pol ε displays a similarly high rate of DNA synthesis (115). However, dNTP levels in the cell are far below the K m values for these enzymes (4, 116).…”

Section: Lagging Strand Replicationmentioning

confidence: 99%

Eukaryotic DNA Replication Fork

Burgers

Kunkel

2017

Annu. Rev. Biochem.

423

407

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This review focuses on the biogenesis and composition of the eukaryotic DNA replication fork, with an emphasis on the enzymes that synthesize DNA and repair discontinuities on the lagging strand of the replication fork. Physical and genetic methodologies aimed at understanding these processes are discussed. The preponderance of evidence supports a model in which DNA polymerase ε (Pol ε) carries out the bulk of leading strand DNA synthesis at an undisturbed replication fork. DNA polymerases α and β carry out the initiation of Okazaki fragment synthesis and its elongation and maturation, respectively. This review also discusses alternative proposals, including cellular processes during which alternative forks may be utilized, and new biochemical studies with purified proteins that are aimed at reconstituting leading and lagging strand DNA synthesis separately and as an integrated replication fork.

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Yeast DNA Polymerase ϵ Catalytic Core and Holoenzyme Have Comparable Catalytic Rates

Cited by 18 publications

References 61 publications

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ

Evidence for the Kinetic Partitioning of Polymerase Activity on G-Quadruplex DNA

Eukaryotic DNA Replication Fork

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