Cells Lacking the PolQ Polymerase Are Moderately Sensitive to Ionizing Radiation and the Oxidant Induced Toxicity of Paraquat and Bleomycin.

Abstract The DNA polymerase POLQ (pol theta) can bypass abasic sites in DNA and thymine glycol, a common product of reactive oxygen species damage to DNA. POLQ may also have a back-up role in base excision repair. To explore the role of POLQ in tolerance of DNA damage following irradiation, and exposure to agents that induce oxidative stress we established bone marrow stromal cell lines from the PolQ+/+ and PolQ−/− mice. Irradiation survival curves were obtained for PolQ +/+, and 2 clones of PolQ −/− cells. Dose-response experiments using the free radical generators paraquat, hydrogen peroxide and bleomycin were done on 2 clones each of PolQ +/+ and PolQ −/− cells. There was no significant difference over 23 weeks in cumulative granulopoiesis in LTBMCs from PolQ +/+ and PolQ −/− mice. PolQ+/+ cells and 2 independent clones of PolQ−/− cells were exposed to 0 – 8 Gy of gamma radiation, and colony forming ability was measured. PolQ −/− cells were more sensitive to irradiation as shown by decreased Do from 1.38 + 0.06 Gy for PolQ +/+ cells compared to 1.27 + 0.16 and 0.98 + 0.10 (p = 0.0316) Gy for PolQ −/− clones 1 and 3 respectively. Micronucleated reticulocytes were measured in peripheral blood of PolQ+/+, +/− or −/− mice after irradiation with 75 cGy or 700 cGy. Before irradiation, PolQ−/− mice had a higher fraction of MN- reticulocytes compared to PolQ+/+. Forty hr after irradiation, MN reticulocytes in PolQ−/− mice increased to 8.53 ± 1.48%, compared to 2.35 ± 0.12% and 2.55 ± 0.17% in PolQ+/+ mice (p = 0.0032). PolQ +/+ and PolQ −/− cells were exposed to paraquat (0–20 mM), hydrogen peroxide (0–40 uM) and bleomycin (0–5 ug/ml) for 1 hr prior to plating. After 7 days, the cells were stained and colonies of 50 cells or greater were counted. Statistical analysis was done using ANCOVA. PolQ −/− cells were modestly more sensitive to paraquat (p< 0.0001) and bleomycin (p< 0.0001) than PolQ +/+ cells. When the cells were treated with hydrogen peroxide, there was no significant difference in surviving fraction (p=0.7327) between the PolQ +/+ and the −/− cells. The moderate but significant sensitivity of POLQ-defective cells to bleomycin, ionizing radiation and paraquat supports the hypothesis that POLQ normally participates in helping cells tolerate DNA damage. The increased frequencies of spontaneous and radiation-induced micronuclei in peripheral blood red cells indicate that POLQ has a role in maintaining genomic integrity via limiting double-strand DNA breaks.

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Poly(ADP-ribose) polymerase in base excision repair: always engaged, but not essential for DNA damage processing.

Acta Biochimica Polonica ◽

10.18388/abp.2003_3724 ◽

2003 ◽

Vol 50 (1) ◽

pp. 169-179 ◽

Cited By ~ 66

Author(s):

Sarah L Allinson ◽

Irina I Dianova ◽

Grigory L Dianov

Keyword(s):

Dna Damage ◽

Base Excision Repair ◽

Excision Repair ◽

Deficient Mouse ◽

Dna Breaks ◽

Abasic Sites ◽

Cell Extracts ◽

Mouse Cells ◽

Base Excision ◽

Parp 1

Poly(ADP-ribose) polymerase (PARP-1) is an abundant nuclear protein with a high affinity for single- and double-strand DNA breaks. Its binding to strand breaks promotes catalysis of the covalent modification of nuclear proteins with poly(ADP-ribose) synthesised from NAD(+). PARP-1-knockout cells are extremely sensitive to alkylating agents, suggesting the involvement of PARP-1 in base excision repair; however, its role remains unclear. We investigated the dependence of base excision repair pathways on PARP-1 and NAD(+) using whole cell extracts derived from normal and PARP-1 deficient mouse cells and DNA substrates containing abasic sites. In normal extracts the rate of repair was highly dependent on NAD(+). We found that in the absence of NAD(+) repair was slowed down 4-6-fold after incision of the abasic site. We also established that in extracts from PARP-1 deficient mouse cells, repair of both regular and reduced abasic sites was increased with respect to normal extracts and was NAD(+)-independent, suggesting that in both short- and long-patch BER PARP-1 slows down, rather than stimulates, the repair reaction. Our data support the proposal that PARP-1 does not play a major role in catalysis of DNA damage processing via either base excision repair pathway.

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Physical and functional interactions between MutY glycosylase homologue (MYH) and checkpoint proteins Rad9–Rad1–Hus1

Biochemical Journal ◽

10.1042/bj20060774 ◽

2006 ◽

Vol 400 (1) ◽

pp. 53-62 ◽

Cited By ~ 61

Author(s):

Guoli Shi ◽

Dau-Yin Chang ◽

Chih-Chien Cheng ◽

Xin Guan ◽

Česlovas Venclovas ◽

...

Keyword(s):

Dna Damage ◽

Ionizing Radiation ◽

Binding Site ◽

Fission Yeast ◽

Base Excision Repair ◽

Excision Repair ◽

Direct Role ◽

Checkpoint Proteins ◽

Functional Interactions ◽

Base Excision

The MYH (MutY glycosylase homologue) increases replication fidelity by removing adenines or 2-hydroxyadenine misincorporated opposite GO (7,8-dihydro-8-oxo-guanine). The 9-1-1 complex (Rad9, Rad1 and Hus1 heterotrimer complex) has been suggested as a DNA damage sensor. Here, we report that hMYH (human MYH) interacts with hHus1 (human Hus1) and hRad1 (human Rad1), but not with hRad9. In addition, interactions between MYH and the 9-1-1 complex, from both the fission yeast Schizosaccharomyces pombe and human cells, are partially interchangeable. The major Hus1-binding site is localized to residues 295–350 of hMYH and to residues 245–293 of SpMYH (S. pombe MYH). Val315 of hMYH and Ile261 of SpMYH play important roles for their interactions with Hus1. hHus1 protein and the 9-1-1 complex of S. pombe can enhance the glycosylase activity of SpMYH. Moreover, the interaction of hMYH–hHus1 is enhanced following ionizing radiation. A significant fraction of the hMYH nuclear foci co-localizes with hRad9 foci in H2O2-treated cells. These results reveal that the 9-1-1 complex plays a direct role in base excision repair.

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DNA Repair of 8-Oxo-7,8-Dihydroguanine Lesions in Porphyromonas gingivalis

Journal of Bacteriology ◽

10.1128/jb.00919-08 ◽

2008 ◽

Vol 190 (24) ◽

pp. 7985-7993 ◽

Cited By ~ 14

Author(s):

Leroy G. Henry ◽

Lawrence Sandberg ◽

Kangling Zhang ◽

Hansel M. Fletcher

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Porphyromonas Gingivalis ◽

Parent Strain ◽

Excision Repair ◽

Periodontal Pocket ◽

Chromosomal Dna ◽

Significant Difference ◽

Nucleotide Excision ◽

Base Excision

ABSTRACT The persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. DNA damage is a major consequence of oxidative stress. Unlike the case for other organisms, our previous report suggests a role for a non-base excision repair mechanism for the removal of 8-oxo-7,8-dihydroguanine (8-oxo-G) in P. gingivalis. Because the uvrB gene is known to be important in nucleotide excision repair, the role of this gene in the repair of oxidative stress-induced DNA damage was investigated. A 3.1-kb fragment containing the uvrB gene was PCR amplified from the chromosomal DNA of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic cassette and used to create a uvrB-deficient mutant by allelic exchange. When plated on brucella blood agar, the mutant strain, designated P. gingivalis FLL144, was similar in black pigmentation and beta-hemolysis to the parent strain. In addition, P. gingivalis FLL144 demonstrated no significant difference in growth rate, proteolytic activity, or sensitivity to hydrogen peroxide from that of the parent strain. However, in contrast to the wild type, P. gingivalis FLL144 was significantly sensitive to UV irradiation. The enzymatic removal of 8-oxo-G from duplex DNA was unaffected by the inactivation of the uvrB gene. DNA affinity fractionation identified unique proteins that preferentially bound to the oligonucleotide fragment carrying the 8-oxo-G lesion. Collectively, these results suggest that the repair of oxidative stress-induced DNA damage involving 8-oxo-G may occur by a still undescribed mechanism in P. gingivalis.

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Abasic sites linked to dUTP incorporation in DNA are a major cause of spontaneous mutations in absence of base excision repair and Rad17–Mec3–Ddc1 (9–1–1) DNA damage checkpoint clamp in Saccharomyces cerevisiae

DNA Repair ◽

10.1016/j.dnarep.2011.12.004 ◽

2012 ◽

Vol 11 (3) ◽

pp. 294-303 ◽

Cited By ~ 8

Author(s):

Ada Collura ◽

Patricia Auffret Van Der Kemp ◽

Serge Boiteux

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Base Excision Repair ◽

Excision Repair ◽

Dna Damage Checkpoint ◽

Spontaneous Mutations ◽

Abasic Sites ◽

Base Excision

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Lesion Recognition and Cleavage of Damage-Containing Quadruplexes and Bulged Structures by DNA Glycosylases

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.595687 ◽

2020 ◽

Vol 8 ◽

Author(s):

Alexandra A. Kuznetsova ◽

Olga S. Fedorova ◽

Nikita A. Kuznetsov

Keyword(s):

Excision Repair ◽

Repair Process ◽

Dna Glycosylase ◽

Promoter Regions ◽

Dna Structures ◽

Thymine Glycol ◽

Abasic Sites ◽

Endonuclease Iii ◽

Human Genes ◽

Base Excision

Human telomeres as well as more than 40% of human genes near the promoter regions have been found to contain the sequence that may form a G-quadruplex structure. Other non-canonical DNA structures comprising bulges, hairpins, or bubbles may have a functionally important role during transcription, replication, or recombination. The guanine-rich regions of DNA are hotspots of oxidation that forms 7,8-dihydro-8-oxoguanine, thymine glycol, and abasic sites: the lesions that are handled by the base excision repair pathway. Nonetheless, the features of DNA repair processes in non-canonical DNA structures are still poorly understood. Therefore, in this work, a comparative analysis of the efficiency of the removal of a damaged nucleotide from various G-quadruplexes and bulged structures was performed using endonuclease VIII-like 1 (NEIL1), human 8-oxoguanine-DNA glycosylase (OGG1), endonuclease III (NTH1), and prokaryotic formamidopyrimidine-DNA glycosylase (Fpg), and endonuclease VIII (Nei). All the tested enzymes were able to cleave damage-containing bulged DNA structures, indicating their important role in the repair process when single-stranded DNA and intermediate non–B-form structures such as bubbles and bulges are formed. Nevertheless, our results suggest that the ability to cleave damaged quadruplexes is an intrinsic feature of members of the H2tH structural family, suggesting that these enzymes can participate in the modulation of processes controlled by the formation of quadruplex structures in genomic DNA.

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Base excision repair of ionizing radiation-induced DNA damage in G1 and G2 cell cycle phases

Cancer Cell International ◽

10.1186/1475-2867-7-15 ◽

2007 ◽

Vol 7 (1) ◽

pp. 15 ◽

Cited By ~ 43

Author(s):

M Ahmad Chaudhry

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Ionizing Radiation ◽

Base Excision Repair ◽

Excision Repair ◽

Radiation Induced ◽

Cell Cycle Phases ◽

Base Excision

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XRCC1 Is Specifically Associated with Poly(ADP-Ribose) Polymerase and Negatively Regulates Its Activity following DNA Damage

Molecular and Cellular Biology ◽

10.1128/mcb.18.6.3563 ◽

1998 ◽

Vol 18 (6) ◽

pp. 3563-3571 ◽

Cited By ~ 606

Author(s):

Murielle Masson ◽

Claude Niedergang ◽

Valérie Schreiber ◽

Sylviane Muller ◽

Josiane Menissier-de Murcia ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Mammalian Cells ◽

Excision Repair ◽

Cell Cycle Checkpoint ◽

Dna Breaks ◽

Protective Function ◽

C Terminus ◽

Base Excision

ABSTRACT Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30 ) is a zinc-finger DNA-binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these breaks, the immediate poly(ADP-ribosyl)ation of nuclear proteins involved in chromatin architecture and DNA metabolism converts DNA damage into intracellular signals that can activate DNA repair programs or cell death options. To have greater insight into the physiological function of this enzyme, we have used the two-hybrid system to find genes encoding proteins putatively interacting with PARP. We have identified a physical association between PARP and the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) in theSaccharomyces cerevisiae system, which was further confirmed to exist in mammalian cells. XRCC1 interacts with PARP by its central region (amino acids 301 to 402), which contains a BRCT (BRCA1 C terminus) module, a widespread motif in DNA repair and DNA damage-responsive cell cycle checkpoint proteins. Overexpression of XRCC1 in Cos-7 or HeLa cells dramatically decreases PARP activity in vivo, reinforcing the potential protective function of PARP at DNA breaks. Given that XRCC1 is also associated with DNA ligase III via a second BRCT module and with DNA polymerase β, our results provide strong evidence that PARP is a member of a BER multiprotein complex involved in the detection of DNA interruptions and possibly in the recruitment of XRCC1 and its partners for efficient processing of these breaks in a coordinated manner. The modular organizations of these interactors, associated with small conserved domains, may contribute to increasing the efficiency of the overall pathway.

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PARP1 Is a TRF2-associated Poly(ADP-Ribose)Polymerase and Protects Eroded Telomeres

Molecular Biology of the Cell ◽

10.1091/mbc.e05-07-0672 ◽

2006 ◽

Vol 17 (4) ◽

pp. 1686-1696 ◽

Cited By ~ 71

Author(s):

Marla Gomez ◽

Jun Wu ◽

Valérie Schreiber ◽

John Dunlap ◽

Françoise Dantzer ◽

...

Keyword(s):

Dna Damage ◽

Excision Repair ◽

Dna Breaks ◽

Telomeric Dna ◽

Telomere Repeat ◽

Damage Repair ◽

Binding Factor ◽

Interphase Cells ◽

End To End ◽

Base Excision

Poly(ADP-ribose)polymerase 1 (PARP1) is well characterized for its role in base excision repair (BER), where it is activated by and binds to DNA breaks and catalyzes the poly(ADP-ribosyl)ation of several substrates involved in DNA damage repair. Here we demonstrate that PARP1 associates with telomere repeat binding factor 2 (TRF2) and is capable of poly(ADP-ribosyl)ation of TRF2, which affects binding of TRF2 to telomeric DNA. Immunostaining of interphase cells or metaphase spreads shows that PARP1 is detected sporadically at normal telomeres, but it appears preferentially at eroded telomeres caused by telomerase deficiency or damaged telomeres induced by DNA-damaging reagents. Although PARP1 is dispensable in the capping of normal telomeres, Parp1 deficiency leads to an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA in primary murine cells after induction of DNA damage. Our results suggest that upon DNA damage, PARP1 is recruited to damaged telomeres, where it can help protect telomeres against chromosome end-to-end fusions and genomic instability.

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