Decreased DNA damage by acid and increased repair of acid-damaged DNA in acid-habituatedEscherichia coli

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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Undamaged DNA Transmits and Enhances DNA Damage Checkpoint Signals in Early Embryos

Molecular and Cellular Biology ◽

10.1128/mcb.00195-07 ◽

2007 ◽

Vol 27 (19) ◽

pp. 6852-6862 ◽

Cited By ~ 15

Author(s):

Aimin Peng ◽

Andrea L. Lewellyn ◽

James L. Maller

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Nuclear Dna ◽

Cell Cycle Checkpoint ◽

Dna Damage Checkpoint ◽

Checkpoint Signaling ◽

Checkpoint Response ◽

Egg Extracts ◽

Cycle Checkpoint ◽

Damaged Dna

ABSTRACT In Xenopus laevis embryos, the midblastula transition (MBT) at the 12th cell division marks initiation of critical developmental events, including zygotic transcription and the abrupt inclusion of gap phases into the cell cycle. Interestingly, although an ionizing radiation-induced checkpoint response is absent in pre-MBT embryos, introduction of a threshold amount of undamaged plasmid or sperm DNA allows a DNA damage checkpoint response to be activated. We show here that undamaged threshold DNA directly participates in checkpoint signaling, as judged by several dynamic changes, including H2AX phosphorylation, ATM phosphorylation and loading onto chromatin, and Chk1/Chk2 phosphorylation and release from nuclear DNA. These responses on physically separate threshold DNA require γ-H2AX and are triggered by an ATM-dependent soluble signal initiated by damaged DNA. The signal persists in egg extracts even after damaged DNA is removed from the system, indicating that the absence of damaged DNA is not sufficient to end the checkpoint response. The results identify a novel mechanism by which undamaged DNA enhances checkpoint signaling and provide an example of how the transition to cell cycle checkpoint activation during development is accomplished by maternally programmed increases in the DNA-to-cytoplasm ratio.

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A CAF-1–PCNA-Mediated Chromatin Assembly Pathway Triggered by Sensing DNA Damage

Molecular and Cellular Biology ◽

10.1128/mcb.20.4.1206-1218.2000 ◽

2000 ◽

Vol 20 (4) ◽

pp. 1206-1218 ◽

Cited By ~ 204

Author(s):

Jonathan G. Moggs ◽

Paola Grandi ◽

Jean-Pierre Quivy ◽

Zophonías O. Jónsson ◽

Ulrich Hübscher ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Single Strand ◽

Chromatin Assembly ◽

Checkpoint Control ◽

Free System ◽

Strand Breaks ◽

Assembly Pathway ◽

Single Strand Breaks ◽

Damaged Dna

ABSTRACT Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA–CAF-1 interaction in the context of DNA damage processing and checkpoint control.

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Recruitment of the Nucleotide Excision Repair Endonuclease XPG to Sites of UV-Induced DNA Damage Depends on Functional TFIIH

Molecular and Cellular Biology ◽

10.1128/mcb.00695-06 ◽

2006 ◽

Vol 26 (23) ◽

pp. 8868-8879 ◽

Cited By ~ 56

Author(s):

Angelika Zotter ◽

Martijn S. Luijsterburg ◽

Daniël O. Warmerdam ◽

Shehu Ibrahim ◽

Alex Nigg ◽

...

Keyword(s):

Dna Damage ◽

Nucleotide Excision Repair ◽

Fluorescent Protein ◽

Core Protein ◽

Excision Repair ◽

Mammalian Cell Lines ◽

Nucleotide Excision ◽

Green Fluorescent ◽

Damaged Dna

ABSTRACT The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3′ side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5′ incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.

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DDB2 Complex-Mediated Ubiquitylation around DNA Damage Is Oppositely Regulated by XPC and Ku and Contributes to the Recruitment of XPA

Molecular and Cellular Biology ◽

10.1128/mcb.01460-09 ◽

2010 ◽

Vol 30 (11) ◽

pp. 2708-2723 ◽

Cited By ~ 42

Author(s):

Arato Takedachi ◽

Masafumi Saijo ◽

Kiyoji Tanaka

Keyword(s):

Dna Damage ◽

Ubiquitin Ligase ◽

Xeroderma Pigmentosum ◽

Excision Repair ◽

Dna Binding Protein ◽

Negative Regulator ◽

Ubiquitin Ligase Activity ◽

Nucleotide Excision ◽

Cullin 4A ◽

Damaged Dna

ABSTRACT UV-damaged-DNA-binding protein (UV-DDB) is a heterodimer comprised of DDB1 and DDB2 and integrated in a complex that includes a ubiquitin ligase component, cullin 4A, and Roc1. Here we show that the ubiquitin ligase activity of the DDB2 complex is required for efficient global genome nucleotide excision repair (GG-NER) in chromatin. Mutant DDB2 proteins derived from xeroderma pigmentosum group E patients are not able to mediate ubiquitylation around damaged sites in chromatin. We also found that CSN, a negative regulator of cullin-based ubiquitin ligases, dissociates from the DDB2 complex when the complex binds to damaged DNA and that XPC and Ku oppositely regulate the ubiquitin ligase activity, especially around damaged sites. Furthermore, the DDB2 complex-mediated ubiquitylation plays a role in recruiting XPA to damaged sites. These findings shed some light on the early stages of GG-NER.

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Human cells enter mitosis with damaged DNA after treatment with pharmacological concentrations of genotoxic agents

Biochemical Journal ◽

10.1042/bj20120385 ◽

2012 ◽

Vol 446 (3) ◽

pp. 373-381 ◽

Cited By ~ 24

Author(s):

Philip M. Kubara ◽

Sophie Kernéis-Golsteyn ◽

Aurélie Studény ◽

Brittany B. Lanser ◽

Laurent Meijer ◽

...

Keyword(s):

Dna Damage ◽

Cellular Response ◽

Experimental System ◽

Human Cells ◽

Cyclin Dependent Kinase ◽

Histone H2ax ◽

Checkpoint Adaptation ◽

Genotoxic Agents ◽

G2 Phase ◽

Damaged Dna

In the present paper, we report that mitosis is a key step in the cellular response to genotoxic agents in human cells. Cells with damaged DNA recruit γH2AX (phosphorylated histone H2AX), phosphorylate Chk1 (checkpoint kinase 1) and arrest in the G2-phase of the cell cycle. Strikingly, nearly all cells escape the DNA damage checkpoint and become rounded, by a mechanism that correlates with Chk1 dephosphorylation. The rounded cells are alive and in mitosis as measured by low phospho-Tyr15 Cdk1 (cyclin-dependent kinase 1), high Cdk activity, active Plk1 (Polo-like kinase 1) and high phospho-histone H3 signals. This phenomenon is independent of the type of DNA damage, but is dependent on pharmacologically relevant doses of genotoxicity. Entry into mitosis is likely to be caused by checkpoint adaptation, and the HT-29 cell-based model provides a powerful experimental system in which to explore its molecular basis. We propose that mitosis with damaged DNA is a biologically significant event because it may cause genomic rearrangement in cells that survive genotoxic damage.

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Nucleotide excision repair–induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response

The Journal of Cell Biology ◽

10.1083/jcb.200902150 ◽

2009 ◽

Vol 186 (6) ◽

pp. 835-847 ◽

Cited By ~ 125

Author(s):

Jurgen A. Marteijn ◽

Simon Bekker-Jensen ◽

Niels Mailand ◽

Hannes Lans ◽

Petra Schwertman ◽

...

Keyword(s):

Dna Damage ◽

Nucleotide Excision Repair ◽

Dna Damage Response ◽

Excision Repair ◽

Strand Break ◽

Epigenetic Mark ◽

Histone H2a ◽

Nucleotide Excision ◽

Damage Response ◽

Damaged Dna

Chromatin modifications are an important component of the of DNA damage response (DDR) network that safeguard genomic integrity. Recently, we demonstrated nucleotide excision repair (NER)–dependent histone H2A ubiquitination at sites of ultraviolet (UV)-induced DNA damage. In this study, we show a sustained H2A ubiquitination at damaged DNA, which requires dynamic ubiquitination by Ubc13 and RNF8. Depletion of these enzymes causes UV hypersensitivity without affecting NER, which is indicative of a function for Ubc13 and RNF8 in the downstream UV–DDR. RNF8 is targeted to damaged DNA through an interaction with the double-strand break (DSB)–DDR scaffold protein MDC1, establishing a novel function for MDC1. RNF8 is recruited to sites of UV damage in a cell cycle–independent fashion that requires NER-generated, single-stranded repair intermediates and ataxia telangiectasia–mutated and Rad3-related protein. Our results reveal a conserved pathway of DNA damage–induced H2A ubiquitination for both DSBs and UV lesions, including the recruitment of 53BP1 and Brca1. Although both lesions are processed by independent repair pathways and trigger signaling responses by distinct kinases, they eventually generate the same epigenetic mark, possibly functioning in DNA damage signal amplification.

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Effect of Tai Chi Exercise on DNA Damage, Antioxidant Enzymes, and Oxidative Stress in Middle-Age Adults

Journal of Physical Activity and Health ◽

10.1123/jpah.6.1.43 ◽

2009 ◽

Vol 6 (1) ◽

pp. 43-54 ◽

Cited By ~ 41

Author(s):

J.A. Goon ◽

A.H. Noor Aini ◽

M. Musalmah ◽

M.Y. Yasmin Anum ◽

W.M. Wan Nazaimoon ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Antioxidant Enzymes ◽

Healthy Aging ◽

Tai Chi ◽

Antioxidant Defenses ◽

Sod Activity ◽

Tai Chi Exercise ◽

Sedentary Subjects ◽

Damaged Dna

Background:The biochemical mechanisms involving oxidative stress to explain the relationship between exercise and healthy aging are still unclear.Methods:Tai Chi participants and matched sedentary volunteers age 45 and above were enrolled. Glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT) activities; levels of DNA damage using the comet assay; and malondialdehyde (MDA) and advanced glycation end products (AGE) were determined at 0, 6, and 12 months.Results:Tai Chi subjects had decreased normal and increased mildly damaged DNA with elevated GPx activity after 6 months (n = 25). Plasma MDA and AGE concentrations decreased significantly after 12 months (n = 15) accompanied by increased SOD activity. This may be attributed to the hormesis effect, whereby mild induction of oxidative stress at the first 6 months of exercise resulted in stimulation of antioxidant defenses. These parameters were unchanged in the sedentary subjects in the first 6 months (n = 27) except for elevated SOD activity. After 12 months, the sedentary subjects (n = 17) had decreased normal DNA and increased severely damaged DNA with unaltered MDA and AGE levels while SOD and GPx activities were significantly elevated.Conclusion:Regular Tai Chi exercise stimulated endogenous antioxidant enzymes and reduced oxidative damage markers.

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Cu,Zn-superoxide dismutase deficiency in mice leads to organ-specific increase in oxidatively damaged DNA and NF-κB1 protein activity.

Acta Biochimica Polonica ◽

10.18388/abp.2010_2447 ◽

2010 ◽

Vol 57 (4) ◽

Cited By ~ 5

Author(s):

Agnieszka Siomek ◽

Kamil Brzoska ◽

Barbara Sochanowicz ◽

Daniel Gackowski ◽

Rafal Rozalski ◽

...

Keyword(s):

Dna Damage ◽

Superoxide Dismutase ◽

Dna Binding ◽

Urinary Excretion ◽

Oxidative Dna Damage ◽

Binding Activity ◽

Wild Type ◽

Dna Binding Activity ◽

Organ Specific ◽

Damaged Dna

Earlier experimental studies have demonstrated that: i) Cu,Zn-superoxide dismutase deficiency leads to oxidative stress and carcinogenesis; ii) dysregulation of NF-κB pathway can mediate a wide variety of diseases, including cancer. Therefore, we decided, for the first time, to examine the level of oxidative DNA damage and the DNA binding activity of NF-κB proteins in SOD1 knockout, heterozygous and wild-type mice. Two kinds of biomarkers of oxidatively damaged DNA: urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA were analysed using HPLC-GC-MS and HPLC-EC. The DNA binding activity of p50 and p65 proteins in a nuclear extracts was assessed using NF-κB p50/p65 EZ-TFA transcription factor assay. These parameters were determined in the brain, liver, kidney and urine of SOD1 knockout, heterozygous and wild-type mice. The level of 8-oxodG in DNA was higher in the liver and kidney of knockout mice than in wild type. No differences were found in urinary excretion of 8-oxoGua and 8-oxodG between wild type and the SOD1-deficient animals. The activity of the p50 protein was higher in the kidneys, but surprisingly not in the livers of SOD1-deficient mice, whereas p65 activity did not show any variability. Our results indicate that in Cu,Zn-SOD-deficient animals the level of oxidative DNA damage and NF-κB1 activity are elevated in certain organs only, which may provide some explanation for organ-specific ROS-induced carcinogenesis.

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