Long‐patch DNA repair synthesis during base excision repair in mammalian cells

Exogenous and endogenous damage to the DNA is inevitable. Several DNA repair pathways including base excision, nucleotide excision, mismatch, homologous and non-homologous recombinations are conserved across all organisms to faithfully maintain the integrity of the genome. The base excision repair (BER) pathway functions to repair single-base DNA lesions and during the process creates the premutagenic apurinic/apyrimidinic (AP) sites. In this review, we discuss the components of the BER pathway in the nematode Caenorhabditis elegans and delineate the different phenotypes caused by the deletion or the knockdown of the respective DNA repair gene, as well as the implications. To date, two DNA glycosylases have been identified in C. elegans, the monofunctional uracil DNA glycosylase-1 (UNG-1) and the bifunctional endonuclease III-1 (NTH-1) with associated AP lyase activity. In addition, the animal possesses two AP endonucleases belonging to the exonuclease-3 and endonuclease IV families and in C. elegans these enzymes are called EXO-3 and APN-1, respectively. In mammalian cells, the DNA polymerase, Pol beta, that is required to reinsert the correct bases for DNA repair synthesis is not found in the genome of C. elegans and the evidence indicates that this role could be substituted by DNA polymerase theta (POLQ), which is known to perform a function in the microhomology-mediated end-joining pathway in human cells. The phenotypes observed by the C. elegans mutant strains of the BER pathway raised many challenging questions including the possibility that the DNA glycosylases may have broader functional roles, as discuss in this review.

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DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.1051 ◽

1993 ◽

Vol 13 (2) ◽

pp. 1051-1058 ◽

Cited By ~ 78

Author(s):

Z Wang ◽

X Wu ◽

E C Friedberg

Keyword(s):

Dna Repair ◽

Dna Polymerase ◽

Base Excision Repair ◽

Excision Repair ◽

Repair Synthesis ◽

Dna Polymerase Epsilon ◽

Nuclear Extracts ◽

Polymerase Epsilon ◽

Base Excision ◽

Dna Repair Synthesis

Base excision repair is an important mechanism for correcting DNA damage produced by many physical and chemical agents. We have examined the effects of the REV3 gene and the DNA polymerase genes POL1, POL2, and POL3 of Saccharomyces cerevisiae on DNA repair synthesis is nuclear extracts. Deletional inactivation of REV3 did not affect repair synthesis in the base excision repair pathway. Repair synthesis in nuclear extracts of pol1, pol2, and pol3 temperature-sensitive mutants was normal at permissive temperatures. However, repair synthesis in pol2 nuclear extracts was defective at the restrictive temperature of 37 degrees C and could be complemented by the addition of purified yeast DNA polymerase epsilon. Repair synthesis in pol1 nuclear extracts was proficient at the restrictive temperature unless DNA polymerase alpha was inactivated prior to the initiation of DNA repair. Thermal inactivation of DNA polymerase delta in pol3 nuclear extracts enhanced DNA repair synthesis approximately 2-fold, an effect which could be specifically reversed by the addition of purified yeast DNA polymerase delta to the extract. These results demonstrate that DNA repair synthesis in the yeast base excision repair pathway is catalyzed by DNA polymerase epsilon but is apparently modulated by the presence of DNA polymerases alpha and delta.

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How are base excision DNA repair pathways deployed in vivo?

F1000Research ◽

10.12688/f1000research.10538.1 ◽

2017 ◽

Vol 6 ◽

pp. 279 ◽

Cited By ~ 3

Author(s):

Upasna Thapar ◽

Bruce Demple

Keyword(s):

Dna Repair ◽

Base Excision Repair ◽

Mammalian Cells ◽

Excision Repair ◽

Biochemical Level ◽

Repair Pathways ◽

Base Excision ◽

Base Excision Dna Repair

Since the discovery of the base excision repair (BER) system for DNA more than 40 years ago, new branches of the pathway have been revealed at the biochemical level by in vitro studies. Largely for technical reasons, however, the confirmation of these subpathways in vivo has been elusive. We review methods that have been used to explore BER in mammalian cells, indicate where there are important knowledge gaps to fill, and suggest a way to address them.

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An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.68 ◽

1994 ◽

Vol 14 (1) ◽

pp. 68-76 ◽

Cited By ~ 321

Author(s):

K W Caldecott ◽

C K McKeown ◽

J D Tucker ◽

S Ljungquist ◽

L H Thompson

Keyword(s):

Dna Repair ◽

Affinity Chromatography ◽

Mammalian Cells ◽

Excision Repair ◽

Affinity Purification ◽

Alkylating Agents ◽

Chinese Hamster ◽

Dna Ligase ◽

Base Excision ◽

Dna Ligase Iii

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

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Incorporation of 5’,8-cyclo-2’deoxyadenosines by DNA repair polymerases via base excision repair

DNA Repair ◽

10.1016/j.dnarep.2021.103258 ◽

2021 ◽

pp. 103258

Author(s):

Pawlos S. Tsegay ◽

Daniela Hernandez ◽

Christopher Brache ◽

Chryssostomos Chatgilialoglu ◽

Marios G. Krokidis ◽

...

Keyword(s):

Dna Repair ◽

Base Excision Repair ◽

Excision Repair ◽

Base Excision

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Expansion of base excision repair compensates for a lack of DNA repair by oxidative dealkylation in budding yeast

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.009813 ◽

2019 ◽

Vol 294 (37) ◽

pp. 13629-13637 ◽

Cited By ~ 2

Author(s):

Suzanne J. Admiraal ◽

Daniel E. Eyler ◽

Michael R. Baldwin ◽

Emily M. Brines ◽

Christopher T. Lohans ◽

...

Keyword(s):

Dna Repair ◽

Base Excision Repair ◽

Excision Repair ◽

Budding Yeast ◽

Base Excision

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Impact of hypoxia on DNA repair and genome integrity

Mutagenesis ◽

10.1093/mutage/gez019 ◽

2019 ◽

Vol 35 (1) ◽

pp. 61-68 ◽

Cited By ~ 5

Author(s):

Alanna R Kaplan ◽

Peter M Glazer

Keyword(s):

Dna Repair ◽

Cancer Progression ◽

Base Excision Repair ◽

Mutation Frequency ◽

Excision Repair ◽

Tumour Microenvironment ◽

Genome Integrity ◽

Future Directions ◽

Homology Directed Repair ◽

Base Excision

Abstract Hypoxia is a hallmark of the tumour microenvironment with profound effects on tumour biology, influencing cancer progression, the development of metastasis and patient outcome. Hypoxia also contributes to genomic instability and mutation frequency by inhibiting DNA repair pathways. This review summarises the diverse mechanisms by which hypoxia affects DNA repair, including suppression of homology-directed repair, mismatch repair and base excision repair. We also discuss the effects of hypoxia mimetics and agents that induce hypoxia on DNA repair, and we highlight areas of potential clinical relevance as well as future directions.

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Dynamic Compartmentalization of Base Excision Repair Proteins in Response to Nuclear and Mitochondrial Oxidative Stress

Molecular and Cellular Biology ◽

10.1128/mcb.01357-08 ◽

2008 ◽

Vol 29 (3) ◽

pp. 794-807 ◽

Cited By ~ 31

Author(s):

Lyra M. Griffiths ◽

Dan Swartzlander ◽

Kellen L. Meadows ◽

Keith D. Wilkinson ◽

Anita H. Corbett ◽

...

Keyword(s):

Oxidative Stress ◽

Dna Damage ◽

Dna Repair ◽

Base Excision Repair ◽

Oxidative Dna Damage ◽

Excision Repair ◽

Intracellular Localization ◽

Genotoxic Stress ◽

Mitochondrial Oxidative Stress ◽

Base Excision

ABSTRACT DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.

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