scholarly journals Multisite SUMOylation restrains DNA polymerase η interactions with DNA damage sites

2020 ◽  
Vol 295 (25) ◽  
pp. 8350-8362 ◽  
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.

scholarly journals DNA damage-specific deubiquitination regulates Rad18 functions to suppress mutagenesis

2014 ◽  
Vol 206 (2) ◽  
pp. 183-197 ◽  
Author(s):  
Michelle K. Zeman ◽  
Jia-Ren Lin ◽  
Raimundo Freire ◽  
Karlene A. Cimprich
Keyword(s):  
Dna Damage ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Lesion Bypass ◽  
Dna Damage Tolerance ◽  
E3 Ligases ◽  
Plant Homeodomain ◽  
In Trans ◽  
Fork Stalling ◽  
Proliferating Cell

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.

scholarly journals Complex Formation with Rev1 Enhances the Proficiency of Saccharomyces cerevisiae DNA Polymerase ζ for Mismatch Extension and for Extension Opposite from DNA Lesions

2006 ◽  
Vol 26 (24) ◽  
pp. 9555-9563 ◽  
Author(s):  
Narottam Acharya ◽  
Robert E. Johnson ◽  
Satya Prakash ◽  
Louise Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Dna Lesions ◽  
Synthetic Activity ◽  
Lesion Bypass ◽  
Dna Lesion ◽  
C Terminus ◽  
Accessory Subunit ◽  
Induced Mutagenesis ◽  
Y Family

ABSTRACT Rev1, a Y family DNA polymerase (Pol) functions together with Polζ, a B family Pol comprised of the Rev3 catalytic subunit and Rev7 accessory subunit, in promoting translesion DNA synthesis (TLS). Extensive genetic studies with Saccharomyces cerevisiae have indicated a requirement of both Polζ and Rev1 for damage-induced mutagenesis, implicating their involvement in mutagenic TLS. Polζ is specifically adapted to promote the extension step of lesion bypass, as it proficiently extends primer termini opposite DNA lesions, and it is also a proficient extender of mismatched primer termini on undamaged DNAs. Since TLS through UV-induced lesions and various other DNA lesions does not depend upon the DNA-synthetic activity of Rev1, Rev1 must contribute to Polζ-dependent TLS in a nonenzymatic way. Here, we provide evidence for the physical association of Rev1 with Polζ and show that this binding is mediated through the C terminus of Rev1 and the polymerase domain of Rev3. Importantly, a rev1 mutant that lacks the C-terminal 72 residues which inactivate interaction with Rev3 exhibits the same high degree of UV sensitivity and defectiveness in UV-induced mutagenesis as that conferred by the rev1Δ mutation. We propose that Rev1 binding to Polζ is indispensable for the targeting of Polζ to the replication fork stalled at a DNA lesion. In addition to this structural role, Rev1 binding enhances the proficiency of Polζ for the extension of mismatched primer termini on undamaged DNAs and for the extension of primer termini opposite DNA lesions.

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

scholarly journals Mechanism of double-base lesion bypass catalyzed by a Y-family DNA polymerase

2008 ◽  
Vol 36 (12) ◽  
pp. 3867-3878 ◽  
Author(s):  
Jessica A. Brown ◽  
Sean A. Newmister ◽  
Kevin A. Fiala ◽  
Zucai Suo
Keyword(s):  
Dna Polymerase ◽  
Lesion Bypass ◽  
Double Base ◽  
Base Lesion ◽  
Y Family

scholarly journals DNA Polymerase and dRP-lyase activities of polymorphic variants of human Pol ι

2021 ◽  
Vol 478 (7) ◽  
pp. 1399-1412
Author(s):  
Evgeniy S. Shilkin ◽  
Anastasia S. Gromova ◽  
Margarita P. Smal ◽  
Alena V. Makarova
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Polymerase Activity ◽  
Altered Expression ◽  
Dna Polymerase Iota ◽  
Nucleotide Incorporation ◽  
Lyase Activity ◽  
Polymorphic Variants ◽  
Y Family

Y-family DNA polymerase iota (Pol ι) is involved in DNA damage response and tolerance. Mutations and altered expression level of POLI gene are linked to a higher incidence of cancer. We biochemically characterized five active site polymorphic variants of human Pol ι: R71G (rs3218778), P118L (rs554252419), I236M (rs3218784), E251K (rs3218783) and P365R (rs200852409). We analyzed fidelity of nucleotide incorporation on undamaged DNA, efficiency and accuracy of DNA damage bypass, as well as 5′-deoxyribophosphate lyase (dRP-lyase) activity. The I236M and P118L variants were indistinguishable from the wild-type Pol ι in activity. The E251K and P365R substitutions altered the spectrum of nucleotide incorporation opposite several undamaged DNA bases. The P365R variant also reduced the dRP-lyase activity and possessed the decreased TLS activity opposite 8-oxo-G. The R71G mutation dramatically affected the catalytic activities of Pol ι. The reduced DNA polymerase activity of the R71G variant correlated with an enhanced fidelity of nucleotide incorporation on undamaged DNA, altered lesion-bypass activity and reduced dRP-lyase activity. Therefore, this amino acid substitution likely alters Pol ι functions in vivo.

scholarly journals Oxidative DNA Damage Bypass in Arabidopsis thaliana Requires DNA Polymerase λ and Proliferating Cell Nuclear Antigen 2

2011 ◽  
Vol 23 (2) ◽  
pp. 806-822 ◽  
Author(s):  
Alessandra Amoroso ◽  
Lorenzo Concia ◽  
Caterina Maggio ◽  
Cécile Raynaud ◽  
Catherine Bergounioux ◽  
...  
Keyword(s):  
Dna Damage ◽  
Arabidopsis Thaliana ◽  
Dna Polymerase ◽  
Proliferating Cell Nuclear Antigen ◽  
Oxidative Dna Damage ◽  
Nuclear Antigen ◽  
Dna Polymerase Λ ◽  
Proliferating Cell

scholarly journals A Single Domain in Human DNA Polymerase ι Mediates Interaction with PCNA: Implications for Translesion DNA Synthesis

2005 ◽  
Vol 25 (3) ◽  
pp. 1183-1190 ◽  
Author(s):  
Lajos Haracska ◽  
Narottam Acharya ◽  
Ildiko Unk ◽  
Robert E. Johnson ◽  
Jerard Hurwitz ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Tight Binding ◽  
Dna Lesions ◽  
Nuclear Antigen ◽  
Binding Motifs ◽  
Translesion Dna Synthesis ◽  
Nucleotide Incorporation ◽  
Y Family ◽  
Stalled Replication Forks ◽  
Proliferating Cell

ABSTRACT DNA polymerases (Pols) of the Y family rescue stalled replication forks by promoting replication through DNA lesions. Humans have four Y family Pols, η, ι, κ, and Rev1, of which Pols η, ι, and κ have been shown to physically interact with proliferating cell nuclear antigen (PCNA) and be functionally stimulated by it. However, in sharp contrast to the large increase in processivity that PCNA binding imparts to the replicative Pol, Polδ, the processivity of Y family Pols is not enhanced upon PCNA binding. Instead, PCNA binding improves the efficiency of nucleotide incorporation via a reduction in the apparent Km for the nucleotide. Here we show that Polι interacts with PCNA via only one of its conserved PCNA binding motifs, regardless of whether PCNA is bound to DNA or not. The mode of PCNA binding by Polι is quite unlike that in Polδ, where multisite interactions with PCNA provide for a very tight binding of the replicating Pol with PCNA. We discuss the implications of these observations for the accuracy of DNA synthesis during translesion synthesis and for the process of Pol exchange at the lesion site.

scholarly journals Phosphorylated Rad18 directs DNA Polymerase η to sites of stalled replication

2010 ◽  
Vol 191 (5) ◽  
pp. 953-966 ◽  
Author(s):  
Tovah A. Day ◽  
Komariah Palle ◽  
Laura R. Barkley ◽  
Naoko Kakusho ◽  
Ying Zou ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Replication Fork ◽  
Genome Stability ◽  
Nuclear Antigen ◽  
Binding Motif ◽  
Polymerase Eta ◽  
Cdc7 Kinase ◽  
Replication Fork Stalling ◽  
Fork Stalling ◽  
Y Family

The E3 ubiquitin ligase Rad18 guides DNA Polymerase eta (Polη) to sites of replication fork stalling and mono-ubiquitinates proliferating cell nuclear antigen (PCNA) to facilitate binding of Y family trans-lesion synthesis (TLS) DNA polymerases during TLS. However, it is unclear exactly how Rad18 is regulated in response to DNA damage and how Rad18 activity is coordinated with progression through different phases of the cell cycle. Here we identify Rad18 as a novel substrate of the essential protein kinase Cdc7 (also termed Dbf4/Drf1-dependent Cdc7 kinase [DDK]). A serine cluster in the Polη-binding motif of Rad18 is phosphorylated by DDK. Efficient association of Rad18 with Polη is dependent on DDK and is necessary for redistribution of Polη to sites of replication fork stalling. This is the first demonstration of Rad18 regulation by direct phosphorylation and provides a novel mechanism for integration of S phase progression with postreplication DNA repair to maintain genome stability.

scholarly journals Mechanism of Abasic Lesion Bypass Catalyzed by a Y-family DNA Polymerase

2007 ◽  
Vol 282 (11) ◽  
pp. 8188-8198 ◽  
Author(s):  
Kevin A. Fiala ◽  
Cameron D. Hypes ◽  
Zucai Suo
Keyword(s):  
Dna Polymerase ◽  
Lesion Bypass ◽  
Y Family

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

2017 ◽  
Vol 114 (11) ◽  
pp. E2205-E2214 ◽  
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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