PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching

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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Non-Recombinogenic Functions of Rad51, BRCA2, and Rad52 in DNA Damage Tolerance

Genes ◽

10.3390/genes12101550 ◽

2021 ◽

Vol 12 (10) ◽

pp. 1550

Author(s):

Félix Prado

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Damage Tolerance ◽

Molecular Switches ◽

Strand Exchange ◽

Template Switching ◽

Replication Forks ◽

Lesion Bypass ◽

Dna Damage Tolerance ◽

Exchange Activity

The DNA damage tolerance (DDT) response is aimed to timely and safely complete DNA replication by facilitating the advance of replication forks through blocking lesions. This process is associated with an accumulation of single-strand DNA (ssDNA), both at the fork and behind the fork. Lesion bypass and ssDNA filling can be performed by translation synthesis (TLS) and template switching mechanisms. TLS uses low-fidelity polymerases to incorporate a dNTP opposite the blocking lesion, whereas template switching uses a Rad51/ssDNA nucleofilament and the sister chromatid to bypass the lesion. Rad51 is loaded at this nucleofilament by two mediator proteins, BRCA2 and Rad52, and these three factors are critical for homologous recombination (HR). Here, we review recent advances showing that Rad51, BRCA2, and Rad52 perform some of these functions through mechanisms that do not require the strand exchange activity of Rad51: the formation and protection of reversed fork structures aimed to bypass blocking lesions, and the promotion of TLS. These findings point to the central HR proteins as potential molecular switches in the choice of the mechanism of DDT.

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DNA damage tolerance in stem cells, ageing, mutagenesis, disease and cancer therapy

Nucleic Acids Research ◽

10.1093/nar/gkz531 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7163-7181 ◽

Cited By ~ 12

Author(s):

Bas Pilzecker ◽

Olimpia Alessandra Buoninfante ◽

Heinz Jacobs

Keyword(s):

Dna Damage ◽

Dna Damage Response ◽

Damage Tolerance ◽

Essential Feature ◽

Dna Lesions ◽

Template Switching ◽

Dna Damage Tolerance ◽

Response Network ◽

Damage Response ◽

Fork Stalling

AbstractThe DNA damage response network guards the stability of the genome from a plethora of exogenous and endogenous insults. An essential feature of the DNA damage response network is its capacity to tolerate DNA damage and structural impediments during DNA synthesis. This capacity, referred to as DNA damage tolerance (DDT), contributes to replication fork progression and stability in the presence of blocking structures or DNA lesions. Defective DDT can lead to a prolonged fork arrest and eventually cumulate in a fork collapse that involves the formation of DNA double strand breaks. Four principal modes of DDT have been distinguished: translesion synthesis, fork reversal, template switching and repriming. All DDT modes warrant continuation of replication through bypassing the fork stalling impediment or repriming downstream of the impediment in combination with filling of the single-stranded DNA gaps. In this way, DDT prevents secondary DNA damage and critically contributes to genome stability and cellular fitness. DDT plays a key role in mutagenesis, stem cell maintenance, ageing and the prevention of cancer. This review provides an overview of the role of DDT in these aspects.

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Mechanisms of DNA Damage Tolerance: Post-Translational Regulation of PCNA

Genes ◽

10.3390/genes10010010 ◽

2018 ◽

Vol 10 (1) ◽

pp. 10 ◽

Cited By ~ 31

Author(s):

Wendy Leung ◽

Ryan Baxley ◽

George-Lucian Moldovan ◽

Anja-Katrin Bielinsky

Keyword(s):

Dna Damage ◽

Translational Regulation ◽

Damage Tolerance ◽

Somatic Hypermutation ◽

Critical Role ◽

Nuclear Antigen ◽

Template Switching ◽

Dna Damage Tolerance ◽

Class Switch ◽

Dna Lesion

DNA damage is a constant source of stress challenging genomic integrity. To ensure faithful duplication of our genomes, mechanisms have evolved to deal with damage encountered during replication. One such mechanism is referred to as DNA damage tolerance (DDT). DDT allows for replication to continue in the presence of a DNA lesion by promoting damage bypass. Two major DDT pathways exist: error-prone translesion synthesis (TLS) and error-free template switching (TS). TLS recruits low-fidelity DNA polymerases to directly replicate across the damaged template, whereas TS uses the nascent sister chromatid as a template for bypass. Both pathways must be tightly controlled to prevent the accumulation of mutations that can occur from the dysregulation of DDT proteins. A key regulator of error-prone versus error-free DDT is the replication clamp, proliferating cell nuclear antigen (PCNA). Post-translational modifications (PTMs) of PCNA, mainly by ubiquitin and SUMO (small ubiquitin-like modifier), play a critical role in DDT. In this review, we will discuss the different types of PTMs of PCNA and how they regulate DDT in response to replication stress. We will also cover the roles of PCNA PTMs in lagging strand synthesis, meiotic recombination, as well as somatic hypermutation and class switch recombination.

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The role of the DNA damage checkpoint in regulation of translesion DNA synthesis

Mutagenesis ◽

10.1093/mutage/gem003 ◽

2007 ◽

Vol 22 (3) ◽

pp. 155-160 ◽

Cited By ~ 3

Author(s):

A. Koren

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Dna Damage Checkpoint ◽

Translesion Dna Synthesis ◽

Translesion Dna

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PCNA Ubiquitylation: Instructive or Permissive to DNA Damage Tolerance Pathways?

Biomolecules ◽

10.3390/biom11101543 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1543

Author(s):

Jun Che ◽

Xin Hong ◽

Hai Rao

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Damage Tolerance ◽

Translesion Synthesis ◽

Dna Lesions ◽

Template Switching ◽

Dna Damage Tolerance ◽

Future Studies ◽

Dna Lesion ◽

Replicative Polymerases

DNA lesions escaping from repair often block the DNA replicative polymerases required for DNA replication and are handled during the S/G2 phases by the DNA damage tolerance (DDT) mechanisms, which include the error-prone translesion synthesis (TLS) and the error-free template switching (TS) pathways. Where the mono-ubiquitylation of PCNA K164 is critical for TLS, the poly-ubiquitylation of the same residue is obligatory for TS. However, it is not known how cells divide the labor between TLS and TS. Due to the fact that the type of DNA lesion significantly influences the TLS and TS choice, we propose that, instead of altering the ratio between the mono- and poly-Ub forms of PCNA, the competition between TLS and TS would automatically determine the selection between the two pathways. Future studies, especially the single integrated lesion “i-Damage” system, would elucidate detailed mechanisms governing the choices of specific DDT pathways.

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Replication intermediate architecture reveals the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells

10.1101/2020.10.12.336107 ◽

2020 ◽

Author(s):

Yann Benureau ◽

Caroline Pouvelle ◽

Eliana Moreira Tavares ◽

Pauline Dupaigne ◽

Emmanuelle Despras ◽

...

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Replication Fork ◽

Damage Tolerance ◽

Dna Lesions ◽

Human Cells ◽

Compensatory Mechanism ◽

Dna Damage Tolerance ◽

Replication Intermediate

AbstractDNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remain elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.

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When DNA Polymerases Multitask: Functions Beyond Nucleotidyl Transfer

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.815845 ◽

2022 ◽

Vol 8 ◽

Author(s):

Denisse Carvajal-Maldonado ◽

Lea Drogalis Beckham ◽

Richard D. Wood ◽

Sylvie Doublié

Keyword(s):

Dna Repair ◽

Dna Replication ◽

Dna Synthesis ◽

Extracurricular Activities ◽

Damage Tolerance ◽

Dna Polymerases ◽

Translesion Dna Synthesis ◽

Dna Strands ◽

Translesion Dna ◽

Central Reaction

DNA polymerases catalyze nucleotidyl transfer, the central reaction in synthesis of DNA polynucleotide chains. They function not only in DNA replication, but also in diverse aspects of DNA repair and recombination. Some DNA polymerases can perform translesion DNA synthesis, facilitating damage tolerance and leading to mutagenesis. In addition to these functions, many DNA polymerases conduct biochemically distinct reactions. This review presents examples of DNA polymerases that carry out nuclease (3ʹ—5′ exonuclease, 5′ nuclease, or end-trimming nuclease) or lyase (5′ dRP lyase) extracurricular activities. The discussion underscores how DNA polymerases have a remarkable ability to manipulate DNA strands, sometimes involving relatively large intramolecular movement.

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