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

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.

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Division of labor of Y-family polymerases in translesion-DNA synthesis for distinct types of DNA damage

PLoS ONE ◽

10.1371/journal.pone.0252587 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252587

Author(s):

Yuriko Inomata ◽

Takuya Abe ◽

Masataka Tsuda ◽

Shunichi Takeda ◽

Kouji Hirota

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Division Of Labor ◽

Double Knockout ◽

Translesion Dna Synthesis ◽

Translesion Dna ◽

Template Dna ◽

Y Family ◽

Higher Sensitivity

Living organisms are continuously under threat from a vast array of DNA-damaging agents, which impact genome DNA. DNA replication machinery stalls at damaged template DNA. The stalled replication fork is restarted via bypass replication by translesion DNA-synthesis polymerases, including the Y-family polymerases Polη, Polι, and Polκ, which possess the ability to incorporate nucleotides opposite the damaged template. To investigate the division of labor among these polymerases in vivo, we generated POLη−/−, POLι−/−, POLκ−/−, double knockout (KO), and triple knockout (TKO) mutants in all combinations from human TK6 cells. TKO cells exhibited a hypersensitivity to ultraviolet (UV), cisplatin (CDDP), and methyl methanesulfonate (MMS), confirming the pivotal role played by these polymerases in bypass replication of damaged template DNA. POLη−/− cells, but not POLι−/− or POLκ−/− cells, showed a strong sensitivity to UV and CDDP, while TKO cells showed a slightly higher sensitivity to UV and CDDP than did POLη−/− cells. On the other hand, TKO cells, but not all single KO cells, exhibited a significantly higher sensitivity to MMS than did wild-type cells. Consistently, DNA-fiber assay revealed that Polη plays a crucial role in bypassing lesions caused by UV-mimetic agent 4-nitroquinoline-1-oxide and CDDP, while all three polymerases play complementary roles in bypassing MMS-induced damage. Our findings indicate that the three Y-family polymerases play distinctly different roles in bypass replication, according to the type of DNA damage generated on the template strand.

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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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Ubiquitin-Binding Motifs in REV1 Protein Are Required for Its Role in the Tolerance of DNA Damage

Molecular and Cellular Biology ◽

10.1128/mcb.01118-06 ◽

2006 ◽

Vol 26 (23) ◽

pp. 8892-8900 ◽

Cited By ~ 143

Author(s):

Caixia Guo ◽

Tie-Shan Tang ◽

Marzena Bienko ◽

Joanne L. Parker ◽

Aleksandra B. Bielen ◽

...

Keyword(s):

Dna Damage ◽

Dna Damage Tolerance ◽

Binding Motifs ◽

C Terminus ◽

Ubiquitin Binding ◽

Replication Foci ◽

Induced Mutagenesis ◽

Y Family ◽

Precise Role

ABSTRACT REV1 protein is a eukaryotic member of the Y family of DNA polymerases involved in the tolerance of DNA damage by replicative bypass. The precise role(s) of REV1 in this process is not known. Here we show, by using the yeast two-hybrid assay and the glutathione S-transferase pull-down assay, that mouse REV1 can physically interact with ubiquitin. The association of REV1 with ubiquitin requires the ubiquitin-binding motifs (UBMs) located at the C terminus of REV1. The UBMs also mediate the enhanced association between monoubiquitylated PCNA and REV1. In cells exposed to UV radiation, the association of REV1 with replication foci is dependent on functional UBMs. The UBMs of REV1 are shown to contribute to DNA damage tolerance and damage-induced mutagenesis in vivo.

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The Roles of UmuD in Regulating Mutagenesis

Journal of Nucleic Acids ◽

10.4061/2010/947680 ◽

2010 ◽

Vol 2010 ◽

pp. 1-12 ◽

Cited By ~ 8

Author(s):

Jaylene N. Ollivierre ◽

Jing Fang ◽

Penny J. Beuning

Keyword(s):

Dna Damage ◽

Protein Interactions ◽

Dna Polymerases ◽

Gene Products ◽

E Coli ◽

Domains Of Life ◽

Induced Mutagenesis ◽

Multiple Conformations ◽

Y Family ◽

Damaged Dna

All organisms are subject to DNA damage from both endogenous and environmental sources. DNA damage that is not fully repaired can lead to mutations. Mutagenesis is now understood to be an active process, in part facilitated by lower-fidelity DNA polymerases that replicate DNA in an error-prone manner. Y-family DNA polymerases, found throughout all domains of life, are characterized by their lower fidelity on undamaged DNA and their specialized ability to copy damaged DNA. TwoE. coliY-family DNA polymerases are responsible for copying damaged DNA as well as for mutagenesis. These DNA polymerases interact with different forms of UmuD, a dynamic protein that regulates mutagenesis. The UmuD gene products, regulated by the SOS response, exist in two principal forms:UmuD2, which prevents mutagenesis, andUmuD2′, which facilitates UV-induced mutagenesis. This paper focuses on the multiple conformations of the UmuD gene products and how their protein interactions regulate mutagenesis.

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Noncognate DNA damage prevents the formation of the active conformation of the Y-family DNA polymerases DinB and DNA polymerase κ

FEBS Journal ◽

10.1111/febs.13304 ◽

2015 ◽

Vol 282 (14) ◽

pp. 2646-2660 ◽

Cited By ~ 8

Author(s):

Philip Nevin ◽

Xueguang Lu ◽

Ke Zhang ◽

John R. Engen ◽

Penny J. Beuning

Keyword(s):

Dna Damage ◽

Dna Polymerase ◽

Dna Polymerases ◽

Active Conformation ◽

Y Family

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Y-family DNA polymerases respond to DNA damage-independent inhibition of replication fork progression

The EMBO Journal ◽

10.1038/sj.emboj.7600986 ◽

2006 ◽

Vol 25 (4) ◽

pp. 868-879 ◽

Cited By ~ 64

Author(s):

Veronica G Godoy ◽

Daniel F Jarosz ◽

Fabianne L Walker ◽

Lyle A Simmons ◽

Graham C Walker

Keyword(s):

Dna Damage ◽

Replication Fork ◽

Dna Polymerases ◽

Replication Fork Progression ◽

Y Family

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Access to PCNA by Srs2 and Elg1 Controls the Choice between Alternative Repair Pathways in Saccharomyces cerevisiae

mBio ◽

10.1128/mbio.00705-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 2

Author(s):

Matan Arbel ◽

Alex Bronstein ◽

Soumitra Sau ◽

Batia Liefshitz ◽

Martin Kupiec

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Damage Tolerance ◽

Dna Polymerases ◽

Strand Transfer ◽

Dna Damage Tolerance ◽

Dna Repair Mechanisms ◽

Dna Damaging Agents ◽

Recombination Protein ◽

Repair Mechanisms

ABSTRACT During DNA replication, stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions. Under these circumstances, the processivity factor PCNA gets ubiquitylated at lysine 164, inducing the DNA damage tolerance (DDT) mechanisms that can bypass lesions encountered during DNA replication. PCNA can also be SUMOylated at the same residue or at lysine 127. Surprisingly, pol30-K164R mutants display a higher degree of sensitivity to DNA-damaging agents than pol30-KK127,164RR strains, unable to modify any of the lysines. Here, we show that in addition to translesion synthesis and strand-transfer DDT mechanisms, an alternative repair mechanism (“salvage recombination”) that copies information from the sister chromatid is repressed by the recruitment of Srs2 to SUMOylated PCNA. Overexpression of Elg1, the PCNA unloader, or of the recombination protein Rad52 allows its activation. We dissect the genetic requirements for this pathway, as well as the interactions between Srs2 and Elg1. IMPORTANCE PCNA, the ring that encircles DNA maintaining the processivity of DNA polymerases, is modified by ubiquitin and SUMO. Whereas ubiquitin is required for bypassing lesions through the DNA damage tolerance (DDT) pathways, we show here that SUMOylation represses another pathway, salvage recombination. The Srs2 helicase is recruited to SUMOylated PCNA and prevents the salvage pathway from acting. The pathway can be induced by overexpressing the PCNA unloader Elg1, or the homologous recombination protein Rad52. Our results underscore the role of PCNA modifications in controlling the various bypass and DNA repair mechanisms.

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