A Reexamination of the Nucleotide Incorporation Fidelity of DNA Polymerases†

Biochemistry ◽  
2002 ◽  
Vol 41 (34) ◽  
pp. 10571-10576 ◽  
Author(s):  
Alexander K. Showalter ◽  
Ming-Daw Tsai
Keyword(s):  
Dna Polymerases ◽  
Nucleotide Incorporation

scholarly journals Plant organellar DNA polymerases paralogs exhibit dissimilar nucleotide incorporation fidelity

FEBS Journal ◽  
2018 ◽  
Vol 285 (21) ◽  
pp. 4005-4018 ◽  
Author(s):  
Víctor M. Ayala‐García ◽  
Noe Baruch‐Torres ◽  
Paola L. García‐Medel ◽  
Luis G. Brieba
Keyword(s):  
Dna Polymerases ◽  
Nucleotide Incorporation ◽  
Organellar Dna

scholarly journals Beyond the Lesion: Back to High Fidelity DNA Synthesis

2022 ◽  
Vol 8 ◽  
Author(s):  
Joseph D. Kaszubowski ◽  
Michael A. Trakselis
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Polymerases ◽  
Translesion Synthesis ◽  
Structural Features ◽  
Dna Bases ◽  
High Fidelity ◽  
Nucleotide Incorporation ◽  
Hand Off ◽  
Enzymatic Mechanisms

High fidelity (HiFi) DNA polymerases (Pols) perform the bulk of DNA synthesis required to duplicate genomes in all forms of life. Their structural features, enzymatic mechanisms, and inherent properties are well-described over several decades of research. HiFi Pols are so accurate that they become stalled at sites of DNA damage or lesions that are not one of the four canonical DNA bases. Once stalled, the replisome becomes compromised and vulnerable to further DNA damage. One mechanism to relieve stalling is to recruit a translesion synthesis (TLS) Pol to rapidly synthesize over and past the damage. These TLS Pols have good specificities for the lesion but are less accurate when synthesizing opposite undamaged DNA, and so, mechanisms are needed to limit TLS Pol synthesis and recruit back a HiFi Pol to reestablish the replisome. The overall TLS process can be complicated with several cellular Pols, multifaceted protein contacts, and variable nucleotide incorporation kinetics all contributing to several discrete substitution (or template hand-off) steps. In this review, we highlight the mechanistic differences between distributive equilibrium exchange events and concerted contact-dependent switching by DNA Pols for insertion, extension, and resumption of high-fidelity synthesis beyond the lesion.

scholarly journals Deoxynucleotide Triphosphate Binding Mode Conserved in Y Family DNA Polymerases

2003 ◽  
Vol 23 (8) ◽  
pp. 3008-3012 ◽  
Author(s):  
Robert E. Johnson ◽  
José Trincao ◽  
Aneel K. Aggarwal ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Dna Polymerase ◽  
Close Contact ◽  
Dna Polymerases ◽  
Binding Mode ◽  
Nucleotide Incorporation ◽  
The Family ◽  
And Function ◽  
High Degree ◽  
Y Family ◽  
Deoxynucleotide Triphosphate

ABSTRACT Although DNA polymerase η (Polη) and other Y family polymerases differ in sequence and function from classical DNA polymerases, they all share a similar right-handed architecture with the palm, fingers, and thumb domains. Here, we examine the role in Saccharomyces cerevisiae Polη of three conserved residues, tyrosine 64, arginine 67, and lysine 279, which come into close contact with the triphosphate moiety of the incoming nucleotide, in nucleotide incorporation. We find that mutational alteration of these residues reduces the efficiency of correct nucleotide incorporation very considerably. The high degree of conservation of these residues among the various Y family DNA polymerases suggests that these residues are also crucial for nucleotide incorporation in the other members of the family. Furthermore, we note that tyrosine 64 and arginine 67 are functionally equivalent to the deoxynucleotide triphosphate binding residues arginine 518 and histidine 506 in T7 DNA polymerase, respectively.

scholarly journals Stimulation of DNA Synthesis Activity of Human DNA Polymerase κ by PCNA

2002 ◽  
Vol 22 (3) ◽  
pp. 784-791 ◽  
Author(s):  
Lajos Haracska ◽  
Ildiko Unk ◽  
Robert E. Johnson ◽  
Barbara B. Phillips ◽  
Jerard Hurwitz ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerases ◽  
Protein A ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Physical Interaction ◽  
Abasic Site ◽  
Nucleotide Incorporation ◽  
Synthesis Activity ◽  
Factor C

ABSTRACT Humans have three DNA polymerases, Polη, Polκ, and Polι, which are able to promote replication through DNA lesions. However, the mechanism by which these DNA polymerases are targeted to the replication machinery stalled at a lesion site has remained unknown. Here, we provide evidence for the physical interaction of human Polκ (hPolκ) with proliferating cell nuclear antigen (PCNA) and show that PCNA, replication factor C (RFC), and replication protein A (RPA) act cooperatively to stimulate the DNA synthesis activity of hPolκ. The processivity of hPolκ, however, is not significantly increased in the presence of these protein factors. The efficiency (V max/K m ) of correct nucleotide incorporation by hPolκ is enhanced ∼50- to 200-fold in the presence of PCNA, RFC, and RPA, and this increase in efficiency is achieved by a reduction in the apparent K m for the nucleotide. Although in the presence of these protein factors, the efficiency of the insertion of an A nucleotide opposite an abasic site is increased ∼40-fold, this reaction still remains quite inefficient; thus, it is unlikely that hPolκ would bypass an abasic site by inserting a nucleotide opposite the site.

scholarly journals Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis

2020 ◽  
pp. jbc.RA120.016489
Author(s):  
Shanzhong Gong ◽  
Serdal Kirmizialtin ◽  
Adrienne Chang ◽  
Joshua E. Mayfield ◽  
Yan Jessie Zhang ◽  
...  
Keyword(s):  
Metal Ions ◽  
Metal Ion ◽  
Dna Polymerases ◽  
Critical Role ◽  
Md Simulations ◽  
Enzyme Analysis ◽  
Hiv Reverse Transcriptase ◽  
Nucleotide Incorporation ◽  
Weak Binding ◽  
Nucleotide Specificity

Magnesium ions play a critical role in catalysis by many enzymes and they contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg2+ions in each step in catalysis have not been resolved. We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme-DNA-Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. Increasing Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold by largely increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV RT binds only two metal ions prior to incorporation of a correct base-pair. MD simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. MD simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions the specificity of DNA polymerases.

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