Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase η

ABSTRACT Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase η (Polη) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Polη are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Polη; in this regard, Polη differs strikingly from classical high-fidelity DNA polymerases.

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Evidence for a Watson-Crick Hydrogen Bonding Requirement in DNA Synthesis by Human DNA Polymerase κ

Molecular and Cellular Biology ◽

10.1128/mcb.25.16.7137-7143.2005 ◽

2005 ◽

Vol 25 (16) ◽

pp. 7137-7143 ◽

Cited By ~ 36

Author(s):

William T. Wolfle ◽

M. Todd Washington ◽

Eric T. Kool ◽

Thomas E. Spratt ◽

Sandra A. Helquist ◽

...

Keyword(s):

Hydrogen Bonding ◽

Dna Synthesis ◽

Active Site ◽

Base Pair ◽

High Efficiency ◽

Dna Lesions ◽

Geometric Constraints ◽

Polymerization Reaction ◽

Nucleotide Incorporation ◽

Low Efficiency

ABSTRACT The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Polη, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Polκ, which differs from Polη in having a higher fidelity and which, unlike Polη, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Polη. We find that an isosteric thymidine analog is replicated with low efficiency by Polκ, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Polη and Polκ rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.

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Role of Hoogsteen Edge Hydrogen Bonding at Template Purines in Nucleotide Incorporation by Human DNA Polymerase ι

Molecular and Cellular Biology ◽

10.1128/mcb.00851-06 ◽

2006 ◽

Vol 26 (17) ◽

pp. 6435-6441 ◽

Cited By ~ 23

Author(s):

Robert E. Johnson ◽

Lajos Haracska ◽

Louise Prakash ◽

Satya Prakash

Keyword(s):

Hydrogen Bonding ◽

Dna Polymerase ◽

Active Site ◽

Dna Polymerases ◽

Human Dna ◽

Nucleotide Incorporation ◽

Template Specificity ◽

Hydrogen Bonding Potential ◽

Dna Polymerase Ι

ABSTRACT Human DNA polymerase ι (Pol ι) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol ι with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol ι active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N6 of an A or the O6 of a G, here we provide evidence that whereas hydrogen bonding at N6 is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O6 is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O6 and N7 hydrogen bonding to DNA synthesis by Pol ι, we have examined its proficiency for replicating through the 6 O-methyl guanine and 8-oxoguanine lesions, which affect the O6 and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O6 is an imperative. The dispensability of N6 hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol ι with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N6 positions of templating A.

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Beyond the Lesion: Back to High Fidelity DNA Synthesis

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.811540 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.22.3.784-791.2002 ◽

2002 ◽

Vol 22 (3) ◽

pp. 784-791 ◽

Cited By ~ 124

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.

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Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid

10.1101/2021.07.13.452224 ◽

2021 ◽

Author(s):

Tyler L Dangerfield ◽

Serdal Kirmizialtin ◽

Kenneth A. Johnson

Keyword(s):

Amino Acid ◽

Dna Polymerase ◽

Active Site ◽

Dna Polymerases ◽

Conformational Dynamics ◽

Unnatural Amino Acid ◽

Structural Basis ◽

High Fidelity ◽

Active Site Residues ◽

Kinetic Measurements

High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension (with the correct nucleotide), the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to greatly exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. We show that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, non-ideal base pairing, and a large increase in the distance from the 3′-OH group of the primer strand to the α-phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high fidelity DNA polymerases.

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Visualizing Rev1 catalyze protein-template DNA synthesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2010484117 ◽

2020 ◽

Vol 117 (41) ◽

pp. 25494-25504

Author(s):

Tyler M. Weaver ◽

Luis M. Cortez ◽

Thu H. Khoang ◽

M. Todd Washington ◽

Pratul K. Agarwal ◽

...

Keyword(s):

Hydrogen Bonds ◽

Structural Changes ◽

Dna Polymerases ◽

Forward Direction ◽

Dna Lesions ◽

Side Chain ◽

Arginine Side Chain ◽

Nucleotide Incorporation ◽

Fork Stalling ◽

Dna Helix

During DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites, as well as minor-groove and exocyclic guanine adducts. Lesion bypass is accomplished using a unique protein-template mechanism in which the templating base is evicted from the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain of Rev1. To understand the protein-template mechanism at an atomic level, we employed a combination of time-lapse X-ray crystallography, molecular dynamics simulations, and DNA enzymology on theSaccharomyces cerevisiaeRev1 protein. We find that Rev1 evicts the templating base from the DNA helix prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, although this is not coupled to large structural changes in Rev1 like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two monophosphates, which drives the reaction in the forward direction and prevents pyrophosphorolysis. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These postcatalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.

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A unique arginine cluster in PolDIP2 enhances nucleotide binding and DNA synthesis by PrimPol

10.1101/2020.05.18.101550 ◽

2020 ◽

Cited By ~ 1

Author(s):

Kazutoshi Kasho ◽

Gorazd Stojkovič ◽

Cristina Velázquez-Ruiz ◽

Maria Isabel Martínez-Jiménez ◽

Timothée Laurent ◽

...

Keyword(s):

Dna Synthesis ◽

Dna Polymerases ◽

Polymerase Activity ◽

Dna Lesions ◽

Replication Forks ◽

Flexible Loop ◽

Nucleotide Incorporation ◽

Dntp Binding ◽

Incorporation Efficiency

ABSTRACTReplication forks often stall at damaged DNA. Resumption of DNA synthesis can occur by replacement of the replicative DNA polymerase with specialized, error-prone translesion DNA polymerases (TLS), that have higher tolerance for damaged substrates. Several of these polymerases (Polλ, Polη and PrimPol) are stimulated in DNA synthesis through interaction with PolDIP2, however the mechanism of this PolDIP2-dependent stimulation is still unclear. Here we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol’s enhanced processivity. PolDIP2 increases PrimPol’s primer-template and dNTP binding affinity, which concomitantly enhances PrimPol’s nucleotide incorporation efficiency. This activity is dependent on a unique arginine cluster in PolDIP2 and could be essential for PrimPol to function in vivo, since the polymerase activity of PrimPol alone is very limited. This mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, could be common to all other PolDIP2-interacting TLS polymerases, i.e. Polλ, Polη, Polζ and REV1, and might be critical for their in vivo function of tolerating DNA lesions at physiological nucleotide concentrations.

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Visualizing Rev1 Catalyze Protein-template DNA Synthesis

10.1101/2020.04.10.036236 ◽

2020 ◽

Author(s):

Tyler M. Weaver ◽

Luis M. Cortez ◽

Thu H. Khoang ◽

M. Todd Washington ◽

Pratul Agarwal ◽

...

Keyword(s):

Hydrogen Bonds ◽

Structural Changes ◽

Dna Polymerases ◽

Forward Direction ◽

Time Lapse ◽

Dna Lesions ◽

Side Chain ◽

Arginine Side Chain ◽

Nucleotide Incorporation ◽

Fork Stalling

AbstractDuring DNA replication, replicative DNA polymerases may encounter DNA lesions, which can stall replication forks. One way to prevent replication fork stalling is through the recruitment of specialized translesion synthesis (TLS) polymerases that have evolved to incorporate nucleotides opposite DNA lesions. Rev1 is a specialized TLS polymerase that bypasses abasic sites as well as minor-groove and exocyclic guanine adducts. It does this by using a unique protein-template mechanism in which the template base is flipped out of the DNA helix and the incoming dCTP hydrogen bonds with an arginine side chain. To observe Rev1 catalysis at the atomic level, we employed time-lapse X-ray crystallography. We found that Rev1 flips out the template base prior to binding the incoming nucleotide. Binding the incoming nucleotide changes the conformation of the DNA substrate to orient it for nucleotidyl transfer, and this is not coupled to large structural changes in the protein like those observed with other DNA polymerases. Moreover, we found that following nucleotide incorporation, Rev1 converts the pyrophosphate product to two mono-phosphates, which drives the reaction in the forward direction. Following nucleotide incorporation, the hydrogen bonds between the incorporated nucleotide and the arginine side chain are broken, but the templating base remains extrahelical. These post-catalytic changes prevent potentially mutagenic processive synthesis by Rev1 and facilitate dissociation of the DNA product from the enzyme.

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A well conserved archaeal B-family polymerase functions as a mismatch and lesion extender

10.1101/2021.09.10.459716 ◽

2021 ◽

Author(s):

Xu Feng ◽

Baochang Zhang ◽

Zhe Gao ◽

Ruyi Xu ◽

Xiaotong Liu ◽

...

Keyword(s):

Dna Polymerase ◽

Dna Polymerases ◽

Dna Lesions ◽

Functional Adaptation ◽

Dna Templates ◽

Sulfolobus Islandicus ◽

Nucleotide Incorporation ◽

Damage Repair ◽

Nucleotide Insertion

ABSTRACTB-family DNA polymerases (PolBs) of different groups are widespread in Archaea and different PolBs often coexist in the same organism. Many of these PolB enzymes remain to be investigated. One of the main groups that are poorly characterized is PolB2 whose members occur in many archaea but are predicted as an inactivated form of DNA polymerase. Herein, Sulfolobus islandicus DNA polymerase 2 (Dpo2), a PolB2 enzyme was expressed in its native host and purified. Characterization of the purified enzyme revealed that the polymerase harbors a robust nucleotide incorporation activity, but devoid of the 3’-5’ exonuclease activity. Enzyme kinetics analyses showed that Dpo2 replicates undamaged DNA templates with high fidelity, which is consistent with its inefficient nucleotide insertion activity opposite different DNA lesions. Strikingly, the polymerase is highly efficient in extending mismatches and mispaired primer termini once a nucleotide is placed opposite a damaged site. Together, these data suggested Dpo2 functions as a mismatch and lesion extender, representing a novel type of PolB that is primarily involved in DNA damage repair in Archaea. Insights were also gained into the functional adaptation of the motif C in the mismatch extension of the B-family DNA polymerases.

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