Kinetic characterization of the polymerase and exonuclease activities of the gene 43 protein of bacteriophage T4

Capson, Todd L.; Peliska, James A.; Kaboord, Barbara Fenn; Frey, Michelle West; Lively, Chris R.; Dahlberg, Michael E.; Benkovic, Stephen J.

doi:10.1021/bi00160a007

Cited by 236 publications

(378 citation statements)

References 49 publications

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“…To examine whether the primer/templates chosen were optimal for nucleotide incorporation by pol ␦ and PCNA, longer primer templates were used (36/48G-mer and 36/60G-mer) and the pre-steadystate kinetic analysis of dCTP incorporation was examined for pol ␦ in the presence of PCNA (results not shown). The longer primer templates decreased both the rate and burst amplitude of nucleotide incorporation by ␦⅐PCNA, as seen previously for bacteriophage T4 polymerase (associated with an increase in nonspecific binding of the polymerase and the DNA) (41). pol ␦ and PCNA are predicted to occupy approximately 20 base pairs on duplex DNA, suggesting that the 24/36-mer used in these studies is sufficient for the accommodation of these proteins (42).…”

supporting

confidence: 75%

Kinetic Analysis of Nucleotide Incorporation by Mammalian DNA Polymerase δ

Einolf¹,

Guengerich

2000

Journal of Biological Chemistry

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The kinetics of nucleotide incorporation into 24/36-mer primer/template DNA by purified fetal calf thymus DNA polymerase (pol) ␦ was examined using steadystate and pre-steady-state kinetics. The role of the pol ␦ accessory protein, proliferating cell nuclear antigen (PCNA), on DNA replication by pol ␦ was also examined by kinetic analysis. The steady-state parameter k cat was similar for pol ␦ in the presence and absence of PCNA (0.36 and 0.30 min ؊1 , respectively); however, the K m for dNTP was 20-fold higher in the absence of PCNA (0.067 versus 1.2 M), decreasing the efficiency of nucleotide insertion. Pre-steady-state bursts of nucleotide incorporation were observed for pol ␦ in the presence and absence of PCNA (rates of polymerization (k pol ) of 1260 and 400 min ؊1 , respectively). The reduction in polymerization rate in the absence of PCNA was also accompanied by a 2-fold decrease in burst amplitude. The steady-state exonuclease rate of pol ␦ was 0.56 min ؊1 (no burst, 10 3 -fold lower than the rate of polymerization). The small phosphorothioate effect of 2 for correct nucleotide incorporation into DNA by pol ␦⅐PCNA indicated that the rate-limiting step in the polymerization cycle occurs prior to phosphodiester bond formation. A K d dNTP value of 0.93 M for pol␦⅐dNTP binding was determined by pre-steady-state kinetics. A 5-fold increase in K d DNA for the pol ␦⅐DNA complex was measured in the absence of PCNA. We conclude that the major replicative mammalian polymerase, pol ␦, exhibits kinetic behavior generally similar to that observed for several prokaryotic model polymerases, particularly a rate-limiting step following product formation in the steady state (dissociation of oligonucleotides) and a rate-limiting step (probably conformational change) preceding phosphodiester bond formation. PCNA appears to affect pol ␦ replication in this model mainly by decreasing the dissociation of the polymerase from the DNA. Polymerase (pol)1 ␦ is the major polymerase involved in chromosomal replication in eukaryotic cells (1, 2). pol ␦ is essential for leading and lagging strand DNA synthesis and has also been shown to be involved in DNA repair processes such as long patch base excision repair, nucleotide excision repair, and mismatch repair (3-6). The other eukaryotic polymerases involved in eukaryotic genomic DNA replication are pol ␣ and pol ⑀ (1). pol ␣ is a moderately processive polymerase that is involved in synthesis of RNA-DNA and DNA-DNA primers on the leading and lagging strand (7). pol ⑀ is required for DNA replication in vivo; however, it is not required for in vitro SV40 DNA replication, supporting the other proposed roles of pol ⑀ in lagging strand synthesis, DNA repair, and cell cycle check point control (6,8,9). pol ␦, therefore, has been identified as the enzyme involved in processive replication of the majority of the eukaryotic genome. Understanding the basic replication reaction by mammalian pol ␦ should aid in the elucidation of the mechanisms underlying the effects of other proteins or DNA struc...

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confidence: 75%

Kinetic Analysis of Nucleotide Incorporation by Mammalian DNA Polymerase δ

Einolf¹,

Guengerich

2000

Journal of Biological Chemistry

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“…The failure of L-dCTP incorporation suggests that either L-dCTP was unable to bind to the binary complex of R517A-DNA and form a ternary complex R517A-DNA-L-dCTP, or the ternary complex was formed but was catalytically inactive. To distinguish between these possibilities, we estimated the binding affinity of L-dCTP through a competition assay (31,32), yielding a K d of 36 μM (SI Appendix, Fig. S8C and Table 1).…”

Section: Structural Basis For Potential Catalytic Pathways Of L-nuclementioning

confidence: 99%

Structural basis for the binding and incorporation of nucleotide analogs with L -stereochemistry by human DNA polymerase λ

Vyas¹,

Zahurancik

Suo

2014

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

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Although lamivudine and emtricitabine, two L-deoxycytidine analogs, have been widely used as antiviral drugs for years, a structural basis for D-stereoselectivity against L-dNTPs, enantiomers of natural nucleotides (D-dNTPs), by any DNA polymerase or reverse transcriptase has not been established due to lack of a ternary structure of a polymerase, DNA, and an incoming L-dNTP. Here, we report 2.10-2.25 Å ternary crystal structures of human DNA polymerase λ, DNA, and L-deoxycytidine 5′-triphosphate (L-dCTP), or the triphosphates of lamivudine ((−)3TC-TP) and emtricitabine ((−)FTC-TP) with four ternary complexes per asymmetric unit. The structures of these 12 ternary complexes reveal that relative to D-deoxycytidine 5′-triphosphate (D-dCTP) in the canonical ternary structure of Polλ-DNA-D-dCTP, L-dCTP, (−)3TC-TP, and (−)FTC-TP all have their ribose rotated by 180°. Among the four ternary complexes with a specific L-nucleotide, two are similar and show that the L-nucleotide forms three Watson-Crick hydrogen bonds with the templating nucleotide dG and adopts a chair-like triphosphate conformation. In the remaining two similar ternary complexes, the L-nucleotide surprisingly interacts with the side chain of a conserved active site residue R517 through one or two hydrogen bonds, whereas the templating dG is anchored by a hydrogen bond with the side chain of a semiconserved residue Y505. Furthermore, the triphosphate of the L-nucleotide adopts an unprecedented N-shaped conformation. Our mutagenic and kinetic studies further demonstrate that the side chain of R517 is critical for the formation of the abovementioned four complexes along proposed catalytic pathways for L-nucleotide incorporation and provide the structural basis for the D-stereoselectivity of a DNA polymerase. (Fig. 1), possess L-stereochemistry. Both lamivudine and emtricitabine have been shown to be more effective in inhibiting HIV-1 RT and less toxic than their enantiomeric D-isomers (1-4). In addition, both lamivudine, a potent inhibitor of hepatitis B virus (HBV) (5), and telbivudine, the L-analog of thymidine, are approved drugs for the treatment of HBV infection, whereas emtricitabine is currently in clinical trials for this purpose (6). These L-nucleoside analogs demonstrate less clinical toxicity than their corresponding D-isomers, likely because human DNA polymerases possess strong D-stereoselectivity by preferentially binding and incorporating D-dNTPs over unnatural nucleotides with L-stereochemistry (L-dNTPs) during DNA synthesis. Surprisingly, a structural basis for the discrimination against L-dNTPs by any DNA polymerase or RT has not been established, although D-stereoselectivity has been successfully explored in antiviral drug development.Despite high clinical efficacy, NRTIs are often associated with various drug toxicities resulting from the inhibition of host DNA polymerases that share a catalytic mechanism akin to HIV-1 RT (7). There are 16 identified human DNA polymerases that belong to A-, B-, X-, and Y-families. NRTI-associated mitoc...

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“…Different rate constants for matched (1 s -1 ) and mismatched 3′-termini of the primers (5 s -1 ) implicated "melting" of DNA duplex as the necessary activation step, that is, the rate-limiting step prior to the fast exonucleolytic cleavage (100 s -1 ). 248 The precise coordination of the substrate in the active site is, at least in the subunit of E. coli Pol III, pH-dependent (see above). 241 In the structure of the subunit -dTMP complex, determined at pH 8.5, the phosphate group of dTMP binds with two oxygen atoms to the metal ions: one bridges the metal ions, and one is terminally bound to the site 2 metal ion (Figures 18 and 19).…”

Section: Catalytic Mechanismmentioning

confidence: 99%