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

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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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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Helicase-Like Transcription Factor HLTF and E3 Ubiquitin Ligase SHPRH Confer DNA Damage Tolerance through Direct Interactions with Proliferating Cell Nuclear Antigen (PCNA)

International Journal of Molecular Sciences ◽

10.3390/ijms21030693 ◽

2020 ◽

Vol 21 (3) ◽

pp. 693 ◽

Cited By ~ 8

Author(s):

Mareike Seelinger ◽

Marit Otterlei

Keyword(s):

Dna Damage ◽

Transcription Factor ◽

Damage Tolerance ◽

Genome Instability ◽

Ring Finger ◽

Nuclear Antigen ◽

Common Mutation ◽

Dna Damage Tolerance ◽

Replicative Stress ◽

Template Switch

To prevent replication fork collapse and genome instability under replicative stress, DNA damage tolerance (DDT) mechanisms have evolved. The RAD5 homologs, HLTF (helicase-like transcription factor) and SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase), both ubiquitin ligases, are involved in several DDT mechanisms; DNA translesion synthesis (TLS), fork reversal/remodeling and template switch (TS). Here we show that these two human RAD5 homologs contain functional APIM PCNA interacting motifs. Our results show that both the role of HLTF in TLS in HLTF overexpressing cells, and nuclear localization of SHPRH, are dependent on interaction of HLTF and SHPRH with PCNA. Additionally, we detected multiple changes in the mutation spectra when APIM in overexpressed HLTF or SHPRH were mutated compared to overexpressed wild type proteins. In plasmids from cells overexpressing the APIM mutant version of HLTF, we observed a decrease in C to T transitions, the most common mutation caused by UV irradiation, and an increase in mutations on the transcribed strand. These results strongly suggest that direct binding of HLTF and SHPRH to PCNA is vital for their function in DDT.

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Srs2 Plays a Critical Role in Reversible G2 Arrest upon Chronic and Low Doses of UV Irradiation via Two Distinct Homologous Recombination-Dependent Mechanisms in Postreplication Repair-Deficient Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00453-10 ◽

2010 ◽

Vol 30 (20) ◽

pp. 4840-4850 ◽

Cited By ~ 15

Author(s):

Takashi Hishida ◽

Yoshihiro Hirade ◽

Nami Haruta ◽

Yoshino Kubota ◽

Hiroshi Iwasaki

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Excision Repair ◽

Critical Role ◽

Nuclear Antigen ◽

Dna Damage Tolerance ◽

Checkpoint Activation ◽

Postreplication Repair ◽

Nucleotide Excision ◽

Proliferating Cell

ABSTRACT Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G2 arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G2 arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G2 arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.

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Monoubiquitylation of histone H2B contributes to the bypass of DNA damage during and after DNA replication

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1612633114 ◽

2017 ◽

Vol 114 (11) ◽

pp. E2205-E2214 ◽

Cited By ~ 19

Author(s):

Shih-Hsun Hung ◽

Ronald P. Wong ◽

Helle D. Ulrich ◽

Cheng-Fu Kao

Keyword(s):

Dna Damage ◽

Replication Fork ◽

Dna Lesions ◽

Template Switching ◽

Lesion Bypass ◽

Dna Damage Tolerance ◽

Cytological Evidence ◽

Histone H2b ◽

Chromatin Dynamics ◽

Dna Lesion

DNA lesion bypass is mediated by DNA damage tolerance (DDT) pathways and homologous recombination (HR). The DDT pathways, which involve translesion synthesis and template switching (TS), are activated by the ubiquitylation (ub) of PCNA through components of the RAD6-RAD18 pathway, whereas the HR pathway is independent of RAD18. However, it is unclear how these processes are coordinated within the context of chromatin. Here we show that Bre1, an ubiquitin ligase specific for histone H2B, is recruited to chromatin in a manner coupled to replication of damaged DNA. In the absence of Bre1 or H2Bub, cells exhibit accumulation of unrepaired DNA lesions. Consequently, the damaged forks become unstable and resistant to repair. We provide physical, genetic, and cytological evidence that H2Bub contributes toward both Rad18-dependent TS and replication fork repair by HR. Using an inducible system of DNA damage bypass, we further show that H2Bub is required for the regulation of DDT after genome duplication. We propose that Bre1-H2Bub facilitates fork recovery and gap-filling repair by controlling chromatin dynamics in response to replicative DNA damage.

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Functional and Physical Interaction of Yeast Mgs1 with PCNA: Impact on RAD6-Dependent DNA Damage Tolerance

Molecular and Cellular Biology ◽

10.1128/mcb.00307-06 ◽

2006 ◽

Vol 26 (14) ◽

pp. 5509-5517 ◽

Cited By ~ 48

Author(s):

Takashi Hishida ◽

Tomoko Ohya ◽

Yoshino Kubota ◽

Yusuke Kamada ◽

Hideo Shinagawa

Keyword(s):

Dna Damage ◽

Posttranslational Modifications ◽

Cell Cycle Progression ◽

Damage Tolerance ◽

Genome Instability ◽

Dna Helicase ◽

Nuclear Antigen ◽

Physical Interaction ◽

Dna Damage Tolerance ◽

Exogenous Dna

ABSTRACT Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.

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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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Control of PCNA deubiquitylation in yeast

Biochemical Society Transactions ◽

10.1042/bst0380104 ◽

2010 ◽

Vol 38 (1) ◽

pp. 104-109 ◽

Cited By ~ 7

Author(s):

Alfonso Gallego-Sánchez ◽

Francisco Conde ◽

Pedro San Segundo ◽

Avelino Bueno

Keyword(s):

Dna Damage ◽

Crucial Role ◽

Proliferating Cell Nuclear Antigen ◽

Molecular Mechanisms ◽

Damage Tolerance ◽

Nuclear Antigen ◽

Dna Damage Tolerance ◽

Sliding Clamp ◽

Evolutionarily Conserved ◽

Proliferating Cell

Eukaryotes ubiquitylate the replication factor PCNA (proliferating-cell nuclear antigen) so that it tolerates DNA damage. Although, in the last few years, the understanding of the evolutionarily conserved mechanism of ubiquitylation of PCNA, and its crucial role in DNA damage tolerance, has progressed impressively, little is known about the deubiquitylation of this sliding clamp in most organisms. In the present review, we will discuss potential molecular mechanisms regulating PCNA deubiquitylation in yeast.

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