DNA damage-specific deubiquitination regulates Rad18 functions to suppress mutagenesis

Deoxyribonucleic acid (DNA) lesions encountered during replication are often bypassed using DNA damage tolerance (DDT) pathways to avoid prolonged fork stalling and allow for completion of DNA replication. Rad18 is a central E3 ubiquitin ligase in DDT, which exists in a monoubiquitinated (Rad18•Ub) and nonubiquitinated form in human cells. We find that Rad18 is deubiquitinated when cells are treated with methyl methanesulfonate or hydrogen peroxide. The ubiquitinated form of Rad18 does not interact with SNF2 histone linker plant homeodomain RING helicase (SHPRH) or helicase-like transcription factor, two downstream E3 ligases needed to carry out error-free bypass of DNA lesions. Instead, it interacts preferentially with the zinc finger domain of another, nonubiquitinated Rad18 and may inhibit Rad18 function in trans. Ubiquitination also prevents Rad18 from localizing to sites of DNA damage, inducing proliferating cell nuclear antigen monoubiquitination, and suppressing mutagenesis. These data reveal a new role for monoubiquitination in controlling Rad18 function and suggest that damage-specific deubiquitination promotes a switch from Rad18•Ub–Rad18 complexes to the Rad18–SHPRH complexes necessary for error-free lesion bypass in cells.

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TheSaccharomyces cerevisiae rev6-1Mutation, Which Inhibits Both the Lesion Bypass and the Recombination Mode of DNA Damage Tolerance, Is an Allele ofPOL30, Encoding Proliferating Cell Nuclear Antigen

Genetics ◽

10.1534/genetics.106.058545 ◽

2006 ◽

Vol 173 (4) ◽

pp. 1983-1989 ◽

Cited By ~ 23

Author(s):

Hengshan Zhang ◽

Peter E. M. Gibbs ◽

Christopher W. Lawrence

Keyword(s):

Dna Damage ◽

Proliferating Cell Nuclear Antigen ◽

Damage Tolerance ◽

Nuclear Antigen ◽

Lesion Bypass ◽

Dna Damage Tolerance ◽

Proliferating Cell

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Multisite SUMOylation restrains DNA polymerase η interactions with DNA damage sites

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013780 ◽

2020 ◽

Vol 295 (25) ◽

pp. 8350-8362 ◽

Cited By ~ 3

Author(s):

Claire Guérillon ◽

Stine Smedegaard ◽

Ivo A. Hendriks ◽

Michael L. Nielsen ◽

Niels Mailand

Keyword(s):

Dna Damage ◽

Dna Polymerase ◽

Feedback Inhibition ◽

Dna Lesions ◽

Nuclear Antigen ◽

Inhibition Mechanism ◽

Proteomic Profiling ◽

Lesion Bypass ◽

Y Family ◽

Damaged Dna

Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.

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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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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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The Role of PCNA Posttranslational Modifications in Translesion Synthesis

Journal of Nucleic Acids ◽

10.4061/2010/761217 ◽

2010 ◽

Vol 2010 ◽

pp. 1-8 ◽

Cited By ~ 14

Author(s):

Montaser Shaheen ◽

Ilanchezhian Shanmugam ◽

Robert Hromas

Keyword(s):

Posttranslational Modifications ◽

Translesion Synthesis ◽

Dna Lesions ◽

Nuclear Antigen ◽

Proliferative Cell Nuclear Antigen ◽

Lesion Bypass ◽

Sumo Ligase ◽

Fork Stalling ◽

The Individual ◽

Template Switch

Organisms are predisposed to different types in DNA damage. Multiple mechanisms have evolved to deal with the individual DNA lesions. Translesion synthesis is a special pathway that enables the replication fork to bypass blocking lesions. Proliferative Cell Nuclear Antigen (PCNA), which is an essential component of the fork, undergoes posttranslational modifications, particularly ubiquitylation and sumoylation that are critical for lesion bypass and for filling of DNA gaps which result from this bypass. A special ubiquitylation system, represented by the Rad6 group of ubiquitin conjugating and ligating enzymes, mediates PCNA mono- and polyubiquitylation in response to fork stalling. The E2 SUMO conjugating enzyme Ubc9 and the E3 SUMO ligase Siz1 are responsible for PCNA sumoylation during undisturbed S phase and in response to fork stalling as well. PCNA monoubiquitylation mediated by Rad6/Rad18 recruits special polymerases to bypass the lesion and fill in the DNA gaps. PCNA polyubiquitylation achieved by ubc13-mms2/Rad 5 in yeast mediates an error-free pathway of lesion bypass likely through template switch. PCNA sumoylation appears required for this error-free pathway, and it plays an antirecombinational role during normal replication by recruiting the helicase Srs2 to prevent sister chromatid exchange and hyper-recombination.

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DNA-damage tolerance through PCNA ubiquitination and sumoylation

Biochemical Journal ◽

10.1042/bcj20190579 ◽

2020 ◽

Vol 477 (14) ◽

pp. 2655-2677

Author(s):

Li Fan ◽

Tonghui Bi ◽

Linxiao Wang ◽

Wei Xiao

Keyword(s):

Dna Damage ◽

Posttranslational Modifications ◽

Damage Tolerance ◽

Nuclear Antigen ◽

Replication Forks ◽

Dna Damage Tolerance ◽

Yeast Saccharomyces Cerevisiae ◽

Dna Damaging Agents ◽

Template Switch ◽

Proliferating Cell

DNA-damage tolerance (DDT) is employed by eukaryotic cells to bypass replication-blocking lesions induced by DNA-damaging agents. In budding yeast Saccharomyces cerevisiae, DDT is mediated by RAD6 epistatic group genes and the central event for DDT is sequential ubiquitination of proliferating cell nuclear antigen (PCNA), a DNA clamp required for replication and DNA repair. DDT consists of two parallel pathways: error-prone DDT is mediated by PCNA monoubiquitination, which recruits translesion synthesis DNA polymerases to bypass lesions with decreased fidelity; and error-free DDT is mediated by K63-linked polyubiquitination of PCNA at the same residue of monoubiquitination, which facilitates homologous recombination-mediated template switch. Interestingly, the same PCNA residue is also subjected to sumoylation, which leads to inhibition of unwanted recombination at replication forks. All three types of PCNA posttranslational modifications require dedicated conjugating and ligation enzymes, and these enzymes are highly conserved in eukaryotes, from yeast to human.

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Conservation of DNA damage tolerance pathways from yeast to humans

Biochemical Society Transactions ◽

10.1042/bst0351334 ◽

2007 ◽

Vol 35 (5) ◽

pp. 1334-1337 ◽

Cited By ~ 19

Author(s):

H.D. Ulrich

Keyword(s):

Damage Tolerance ◽

Reference Model ◽

Current Knowledge ◽

Dna Lesions ◽

Nuclear Antigen ◽

Lesion Bypass ◽

Dna Damage Tolerance ◽

Experimental Systems ◽

Species Specific ◽

Enzymatic Machinery

Damage tolerance mechanisms, which allow the bypass of DNA lesions during replication, are controlled in eukaryotic cells by mono- and poly-ubiquitination of the DNA polymerase cofactor PCNA (proliferating-cell nuclear antigen). In the present review, I will summarize our current knowledge of the enzymatic machinery for ubiquitination of PCNA and the way in which the modifications affect PCNA function during replication and lesion bypass in different organisms. Using the budding yeast as a reference model, I will highlight some of the species-specific differences, but also point out the common principles that emerge from the genetic and biochemical studies of damage tolerance in a range of experimental systems.

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