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

2003 ◽  
Vol 23 (14) ◽  
pp. 5107-5112 ◽  
Author(s):  
M. Todd Washington ◽  
Sandra A. Helquist ◽  
Eric T. Kool ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Hydrogen Bonding ◽  
Hydrogen Bonds ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Dna Lesions ◽  
Geometric Constraints ◽  
High Fidelity ◽  
Nucleotide Incorporation

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.

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.

Abstract 4089: Investigating translesion synthesis DNA polymerases roles on tumor protection after temozolomide induced DNA damage

2020 ◽  
Author(s):  
Marcela Teatin Latancia ◽  
André Uchimura Bastos ◽  
Natália Cestari Moreno ◽  
Davi Jardim ◽  
Clarissa RR Rocha ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Polymerases ◽  
Translesion Synthesis ◽  
Tumor Protection

scholarly journals Mechanistic Studies with DNA Polymerases Reveal Complex Outcomes following Bypass of DNA Damage

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Robert L. Eoff ◽  
Jeong-Yun Choi ◽  
F. Peter Guengerich
Keyword(s):  
Dna Damage ◽  
Dna Polymerases ◽  
Structural Features ◽  
Covalent Modification ◽  
Cellular Systems ◽  
Dna Damage Tolerance ◽  
Covalent Modifications ◽  
Template Dna ◽  
Cellular Dysfunction ◽  
Y Family

DNA is a chemically reactive molecule that is subject to many different covalent modifications from sources that are both endogenous and exogenous in origin. The inherent instability of DNA is a major obstacle to genomic maintenance and contributes in varying degrees to cellular dysfunction and disease in multi-cellular organisms. Investigations into the chemical and biological aspects of DNA damage have identified multi-tiered and overlapping cellular systems that have evolved as a means of stabilizing the genome. One of these pathways supports DNA replication events by in a sense adopting the mantra that one must “make the best of a bad situation” and tolerating covalent modification to DNA through less accurate copying of the damaged region. Part of this so-called DNA damage tolerance pathway involves the recruitment of specialized DNA polymerases to sites of stalled or collapsed replication forks. These enzymes have unique structural and functional attributes that often allow bypass of adducted template DNA and successful completion of genomic replication. What follows is a selective description of the salient structural features and bypass properties of specialized DNA polymerases with an emphasis on Y-family members.

scholarly journals Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase in replicating past an abasic site

2007 ◽  
Vol 402 (2) ◽  
pp. 321-329 ◽  
Author(s):  
Giuseppina Blanca ◽  
Emmanuelle Delagoutte ◽  
Nicolas Tanguy le gac ◽  
Neil P. Johnson ◽  
Giuseppe Baldacci ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Bacteriophage T4 ◽  
Dna Polymerases ◽  
Accessory Proteins ◽  
Clamp Loader ◽  
Abasic Site ◽  
Translesion Dna Synthesis ◽  
Translesion Dna

Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3′–5′ exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3′–5′ exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo− polymerase (T4 DNA polymerase deficient in the 3′–5′ exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo− polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis.

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

2020 ◽  
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.

scholarly journals An archaeal primase functions as a nanoscale caliper to define primer length

2018 ◽  
Vol 115 (26) ◽  
pp. 6697-6702 ◽  
Author(s):  
Jiangyu Yan ◽  
Sandro Holzer ◽  
Luca Pellegrini ◽  
Stephen D. Bell
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
De Novo ◽  
Dna Polymerases ◽  
Measuring System ◽  
Structural Homology ◽  
Dna Primase ◽  
Dna And Rna ◽  
Hand Off ◽  
Rna And Dna

The cellular replicative DNA polymerases cannot initiate DNA synthesis without a priming 3′ OH. During DNA replication, this is supplied in the context of a short RNA primer molecule synthesized by DNA primase. The primase of archaea and eukaryotes, despite having varying subunit compositions, share sequence and structural homology. Intriguingly, archaeal primase has been demonstrated to possess the ability to synthesize DNA de novo, a property shared with the eukaryotic PrimPol enzymes. The dual RNA and DNA synthetic capabilities of the archaeal DNA primase have led to the proposal that there may be a sequential hand-off between these synthetic modes of primase. In the current work, we dissect the functional interplay between DNA and RNA synthetic modes of primase. In addition, we determine the key determinants that govern primer length definition by the archaeal primase. Our results indicate a primer measuring system that functions akin to a caliper.

scholarly journals Genetic and physical interactions between Polη and Rev1 in response to UV-induced DNA damage in mammalian cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tonghui Bi ◽  
Xiaohong Niu ◽  
Chunping Qin ◽  
Wei Xiao
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Damage Tolerance ◽  
Dna Polymerases ◽  
Variant Form ◽  
Physical Interaction ◽  
Uv Damage ◽  
Translesion Dna Synthesis ◽  
Physical Interactions

AbstractIn response to UV irradiation, translesion DNA synthesis (TLS) utilizes specialized DNA polymerases to bypass replication-blocking lesions. In a well-established polymerase switch model, Polη is thought to be a preferred TLS polymerase to insert correct nucleotides across from the thymine dimer, and Rev1 plays a scaffold role through physical interaction with Polη and the Rev7 subunit of Polζ for continual DNA synthesis. Defective Polη causes a variant form of xeroderma pigmentosum (XPV), a disease with predisposition to sunlight-induced skin cancer. Previous studies revealed that expression of Rev1 alone is sufficient to confer enhanced UV damage tolerance in mammalian cells, which depends on its physical interaction with Polζ but is independent of Polη, a conclusion that appears to contradict current literature on the critical roles of Polη in TLS. To test a hypothesis that the Rev1 catalytic activity is required to backup Polη in TLS, we found that the Rev1 polymerase-dead mutation is synergistic with either Polη mutation or the Polη-interaction mutation in response to UV-induced DNA damage. On the other hand, functional complementation of polH cells by Polη relies on its physical interaction with Rev1. Hence, our studies reveal critical interactions between Rev1 and Polη in response to UV damage.

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