Pre-Steady State Kinetic Studies of the Fidelity of Nucleotide Incorporation by Yeast DNA Polymerase δ

Abstract

Eukaryotic DNA polymerase delta (pol δ) is a member of the B family of polymerases and synthesizes most of the lagging strand during DNA replication. Yeast pol δ is a heterotrimer comprised of three subunits: the catalytic subunit (Pol3) and two accessory subunits (Pol31 and Pol32). Although it is one of the major eukaryotic replicative polymerase, the mechanism by which it incorporates nucleotides is unknown. Here we report both steady state and pre-steady state kinetic studies of the fidelity of pol δ. We found that pol δ incorporates nucleotides with an error frequency of 10 −4 to 10 −5 . Furthermore, we showed that for correct versus incorrect nucleotide incorporation, there are significant differences between both pre-steady state kinetic parameters (apparent K d dNTP and k pol ). Somewhat surprisingly, we found that pol δ synthesizes DNA at a slow rate with a k pol of ~1 s −1 . We suggest that, unlike its prokaryotic counterparts, pol δ requires replication accessory factors like proliferating cell nuclear antigen to achieve rapid rates of nucleotide incorporation.Eukaryotic DNA polymerase δ (pol δ), a member of the B family of DNA polymerases, is responsible for synthesizing the bulk of the lagging strand of genomic DNA during normal replication [1][2][3]. In addition, pol δ participates in nucleotide excision repair, base excision repair, and double strand break repair [4]. It also plays an important role in maintaining genome stability, as mutations in pol δ cause an increased frequency of cancer in mice [5][6][7] and have been found in cell lines derived from human cancers [8,9].Yeast pol δ is a heterotrimer, comprised of three subunits: Pol3 (125 kDa), Pol31 (55 kDa), and Pol32 (40 kDa) [10]. Pol3 is the catalytic subunit and possesses both DNA polymerase and 3′-5′ exonuclease catalytic activities [11,12]. Pol31 is an accessory subunit that is essential for viability [13]. By contrast, Pol32 is an accessory subunit that is not essential for viability; however, cells lacking this subunit show defects in DNA replication and repair [13]. Pol32 also contains the consensus proliferating cell nuclear antigen (PCNA) binding motif, and interactions with PCNA significantly increase the processivity of pol δ [14,15].The structure of the Pol3 catalytic subunit of pol δ bound to both DNA and incoming dNTP substrates has recently been determined [16]. Overall, the structure of the Pol3 subunit resembles that of the bacteriophage RB69 DNA polymerase, another member of the B † The project described was supported by Award Number GM081433 from the National Institute of General Medical Sciences to M.T.W and Award Number CA138546 from the National Cancer Institute to S.P. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences, the National Cancer Institute, or the National Institutes of Health. * To whom correspondence should be addressed: M. Todd Washington, Department of Biochemistry, 4-403 Bowen Scienc...

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

“…It was previously reported that yeast Pol ␦ has an apparent k pol of about 1 s Ϫ1 for the chemical step, which is too slow, considering that the fork movement has been estimated to be ϳ100 nucleotides s Ϫ1 (51,52 (15,31). The k pol and K d dTTP of Pol ⑀ are also similar to k pol values reported for other polymerases, such as T4, T7, and RB69 gp43 (33,40,53) (Table 5).…”

Kinetics and thermodynamics of exonuclease-deficient DNA polymerases

“…Indeed, the discrimination between correct and incorrect nucleotides may already be quite efficient without dedicated proofreading mechanisms. By explicitly taking into account the dependence of the rates on the concentrations of nucleotides and pyrophosphate, the theory provides direct comparison with experimental observations [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31]. In particular, the theory explains the Michaelis-Menten dependence of the mean growth velocity on the nucleotide concentration, which is a basic feature of enzymatic kinetics.…”

“…Although the first assumption is not supported by experimental observations [19], it is often considered because of its great simplicity. The second assumption captures the observation that the polymerization rate constants k (13) take similar values within the set of correct (respectively incorrect) pairings [13,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31]. According to these assumptions, the model only needs the four rate constants k p ±c and k p ±i for polymerization and depolymerization, together with the two Michaelis-Menten constants K c and K i for correct and incorrect pairings.…”

Eukaryotic DNA Replication Fork

“…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). When measured at physiological dNTP levels, and in the presence of competing rNTPs, DNA synthesis proceeds at ~50 nt/sec, which is commensurate with rates of fork movement in the cell (117).…”