scholarly journals ATR-mediated phosphorylation of DNA polymerase η is needed for efficient recovery from UV damage

2011 ◽  
Vol 192 (2) ◽  
pp. 219-227 ◽  
Author(s):  
Thomas Göhler ◽  
Simone Sabbioneda ◽  
Catherine M. Green ◽  
Alan R. Lehmann
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Mammalian Cells ◽  
Translesion Synthesis ◽  
Physical Interaction ◽  
Uv Damage ◽  
Checkpoint Response ◽  
Ubiquitin Binding Domain ◽  
Efficient Recovery ◽  
Y Family

DNA polymerase η (polη) belongs to the Y-family of DNA polymerases and facilitates translesion synthesis past UV damage. We show that, after UV irradiation, polη becomes phosphorylated at Ser601 by the ataxia-telangiectasia mutated and Rad3-related (ATR) kinase. DNA damage–induced phosphorylation of polη depends on its physical interaction with Rad18 but is independent of PCNA monoubiquitination. It requires the ubiquitin-binding domain of polη but not its PCNA-interacting motif. ATR-dependent phosphorylation of polη is necessary to restore normal survival and postreplication repair after ultraviolet irradiation in xeroderma pigmentosum variant fibroblasts, and is involved in the checkpoint response to UV damage. Taken together, our results provide evidence for a link between DNA damage–induced checkpoint activation and translesion synthesis in mammalian cells.

scholarly journals Genetic and physical interactions between Polη and Rev1 in response to UV-induced DNA damage in mammalian cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tonghui Bi ◽  
Xiaohong Niu ◽  
Chunping Qin ◽  
Wei Xiao
Keyword(s):  
Dna Damage ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Damage Tolerance ◽  
Dna Polymerases ◽  
Variant Form ◽  
Physical Interaction ◽  
Uv Damage ◽  
Translesion Dna Synthesis ◽  
Physical Interactions

AbstractIn response to UV irradiation, translesion DNA synthesis (TLS) utilizes specialized DNA polymerases to bypass replication-blocking lesions. In a well-established polymerase switch model, Polη is thought to be a preferred TLS polymerase to insert correct nucleotides across from the thymine dimer, and Rev1 plays a scaffold role through physical interaction with Polη and the Rev7 subunit of Polζ for continual DNA synthesis. Defective Polη causes a variant form of xeroderma pigmentosum (XPV), a disease with predisposition to sunlight-induced skin cancer. Previous studies revealed that expression of Rev1 alone is sufficient to confer enhanced UV damage tolerance in mammalian cells, which depends on its physical interaction with Polζ but is independent of Polη, a conclusion that appears to contradict current literature on the critical roles of Polη in TLS. To test a hypothesis that the Rev1 catalytic activity is required to backup Polη in TLS, we found that the Rev1 polymerase-dead mutation is synergistic with either Polη mutation or the Polη-interaction mutation in response to UV-induced DNA damage. On the other hand, functional complementation of polH cells by Polη relies on its physical interaction with Rev1. Hence, our studies reveal critical interactions between Rev1 and Polη in response to UV damage.

scholarly journals Replication Past the -Radiation-Induced Guanine-Thymine Cross-Link G[8,5-Me]T by Human and Yeast DNA Polymerase

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Paromita Raychaudhury ◽  
Ashis K. Basu
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Translesion Synthesis ◽  
Kidney Cells ◽  
Equal Frequency ◽  
Cross Link ◽  
Dominant Mutation ◽  
Human Embryonic Kidney Cells ◽  
Radiation Induced

-Radiation-induced intrastrand guanine-thymine cross-link, G[8,5-Me]T, hinders replicationin vitroand is mutagenic in mammalian cells. Herein we reportin vitrotranslesion synthesis of G[8,5-Me]T by human and yeast DNA polymerase (hPol and yPol ). dAMP misincorporation opposite the cross-linked G by yPol was preferred over correct incorporation of dCMP, but further extension was 100-fold less efficient for :A compared to :C. For hPol , both incorporation and extension were more efficient with the correct nucleotides. To evaluate translesion synthesis in the presence of all four dNTPs, we have developed a plasmid-based DNA sequencing assay, which showed that yPol was more error-prone. Mutational frequencies of yPol and hPol were 36% and 14%, respectively. Targeted was the dominant mutation by both DNA polymerases. But yPol induced targeted in 23% frequency relative to 4% by hPol . For yPol , targeted and constituted 83% of the mutations. By contrast, with hPol , semi-targeted mutations (7.2%), that is, mutations at bases near the lesion, occurred at equal frequency as the targeted mutations (6.9%). The kind of mutations detected with hPol showed significant similarities with the mutational spectrum of G[8,5-Me]T in human embryonic kidney cells.

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 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 Y-family DNA polymerase-independent gap-filling translesion synthesis across aristolochic acid-derived adenine adducts in mouse cells

DNA Repair ◽  
2016 ◽  
Vol 46 ◽  
pp. 55-60 ◽  
Author(s):  
Keiji Hashimoto ◽  
Radha Bonala ◽  
Francis Johnson ◽  
Arthur P. Grollman ◽  
Masaaki Moriya
Keyword(s):  
Dna Polymerase ◽  
Aristolochic Acid ◽  
Translesion Synthesis ◽  
Gap Filling ◽  
Mouse Cells ◽  
Adenine Adducts ◽  
Y Family

scholarly journals Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance

2019 ◽  
Author(s):  
Anna Fortuny ◽  
Audrey Chansard ◽  
Pierre Caron ◽  
Odile Chevallier ◽  
Olivier Leroy ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Fate ◽  
Histone Modifications ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Molecular Mechanisms ◽  
Transcriptional Silencing ◽  
Genotoxic Stress ◽  
Higher Order ◽  
Uv Damage

ABSTRACTHeterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a system for targeting UV damage to pericentric heterochromatin in mammalian cells and for tracking the heterochromatin response to UV in real time. We uncover profound heterochromatin compaction changes during repair, orchestrated by the UV damage sensor DDB2, which stimulates linker histone displacement from chromatin. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We unveil a central role for the methyltransferase SETDB1 in the maintenance of heterochromatic histone marks after UV, SETDB1 coordinating histone methylation with new histone deposition in damaged heterochromatin, thus protecting cells from genome instability. Our data shed light on fundamental molecular mechanisms safeguarding higher-order chromatin integrity following DNA damage.

scholarly journals Imaging the response to DNA damage in heterochromatin domains reveals core principles of heterochromatin maintenance

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anna Fortuny ◽  
Audrey Chansard ◽  
Pierre Caron ◽  
Odile Chevallier ◽  
Olivier Leroy ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Fate ◽  
Histone Modifications ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Molecular Mechanisms ◽  
Transcriptional Silencing ◽  
Genotoxic Stress ◽  
Higher Order ◽  
Uv Damage

AbstractHeterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a system for targeting UV damage to pericentric heterochromatin in mammalian cells and for tracking the heterochromatin response to UV in real time. We uncover profound heterochromatin compaction changes during repair, orchestrated by the UV damage sensor DDB2, which stimulates linker histone displacement from chromatin. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We unveil a central role for the methyltransferase SETDB1 in the maintenance of heterochromatic histone marks after UV. SETDB1 coordinates histone methylation with new histone deposition in damaged heterochromatin, thus protecting cells from genome instability. Our data shed light on fundamental molecular mechanisms safeguarding higher-order chromatin integrity following DNA damage.

Inhibition of S-phase progression triggered by UVA-induced ROS does not require a functional DNA damage checkpoint response in mammalian cells

DNA Repair ◽  
2008 ◽  
Vol 7 (9) ◽  
pp. 1500-1516 ◽  
Author(s):  
Pierre-Marie Girard ◽  
Mariaelena Pozzebon ◽  
Fabien Delacôte ◽  
Thierry Douki ◽  
Violetta Smirnova ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
S Phase ◽  
Dna Damage Checkpoint ◽  
Checkpoint Response ◽  
Phase Progression ◽  
Functional Dna
Sign in / Sign up

Export Citation Format

Share Document