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

1994 ◽  
Vol 14 (1) ◽  
pp. 68-76 ◽  
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.

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

1994 ◽  
Vol 14 (1) ◽  
pp. 68-76
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.

scholarly journals Involvement of XRCC1 and DNA Ligase III Gene Products in DNA Base Excision Repair

1997 ◽  
Vol 272 (38) ◽  
pp. 23970-23975 ◽  
Author(s):  
Enrico Cappelli ◽  
Richard Taylor ◽  
Michela Cevasco ◽  
Angelo Abbondandolo ◽  
Keith Caldecott ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Ligase ◽  
Gene Products ◽  
Dna Base Excision Repair ◽  
Dna Base ◽  
Base Excision ◽  
Dna Ligase Iii

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

EMBO Reports ◽  
2003 ◽  
Vol 4 (4) ◽  
pp. 363-367 ◽  
Author(s):  
Ulrike Sattler ◽  
Philippe Frit ◽  
Bernard Salles ◽  
Patrick Calsou
Keyword(s):  
Dna Repair ◽  
Base Excision Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Repair Synthesis ◽  
Base Excision ◽  
Dna Repair Synthesis

scholarly journals Repair of DNA alkylation damage.

1998 ◽  
Vol 45 (1) ◽  
pp. 191-202 ◽  
Author(s):  
M Bouziane ◽  
F Miao ◽  
N Ye ◽  
G Holmquist ◽  
G Chyzak ◽  
...  
Keyword(s):  
Dna Damage ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Phosphoglycerate Kinase ◽  
Amino Acid Sequences ◽  
Dna Glycosylases ◽  
Alkylation Damage ◽  
Base Excision ◽  
Methylating Agents

Alkylation damage of DNA is one of the major types of insults which cells must repair to remain viable. One way alkylation damaged ring nitrogens are repaired is via the Base Excision Repair (BER) pathway. Examination of mutants in both BER and Nucleotide Excision Repair show that there is actually an overlap of repair by these two pathways for the removal of cytotoxic lesions in Escherichia coli. The enzymes removing damaged bases in the first step in the BER pathway are DNA glycosylases. The coding sequences for a number of methylpurine-DNA glycosylases (MPG proteins) were cloned, and a comparison of the amino-acid sequences shows that there are some similarities between these proteins, but nonetheless, compared to other DNA glycosylases, MPG proteins are more divergent. MPG proteins have been purified to homogeneity and used to identify their substrates ranging from methylating agents to deamination products to oxidatively damaged bases. The ligation-mediated polymerase chain reaction has been used to study the formation of alkylation damage, and its repair in mammalian cells. We have studied DNA damage in the PGK1 gene for a series of DNA alkylating agents including N-methyl-N'-nitro-N-nitrosoguanidine, Mechlorethamine, and Chlorambucil and shown that the damage observed in the PGK1 (phosphoglycerate kinase 1) gene depends on the alkylating agent used. This report reviews the literature on the MPG proteins, DNA glycosylases removing 3-methyladenine, and the use of these enzymes to detect DNA damage at the nucleotide level.

scholarly journals How are base excision DNA repair pathways deployed in vivo?

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 279 ◽  
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.

scholarly journals Overexpression of DNA ligase III in mitochondria protects cells against oxidative stress and improves mitochondrial DNA base excision repair

DNA Repair ◽  
2014 ◽  
Vol 16 ◽  
pp. 44-53 ◽  
Author(s):  
Mansour Akbari ◽  
Guido Keijzers ◽  
Scott Maynard ◽  
Morten Scheibye-Knudsen ◽  
Claus Desler ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Mitochondrial Dna ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Ligase ◽  
Dna Base Excision Repair ◽  
Dna Base ◽  
Base Excision ◽  
Dna Ligase Iii

scholarly journals Nucleosome Disruption by DNA Ligase III-XRCC1 Promotes Efficient Base Excision Repair

2011 ◽  
Vol 31 (22) ◽  
pp. 4623-4632 ◽  
Author(s):  
I. D. Odell ◽  
J.-E. Barbour ◽  
D. L. Murphy ◽  
J. A. Della-Maria ◽  
J. B. Sweasy ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Ligase ◽  
Base Excision ◽  
Dna Ligase Iii

Preferential repair of damage in actively transcribed DNA sequences in vivo

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 605-611 ◽  
Author(s):  
Philip C. Hanawalt
Keyword(s):  
Dna Repair ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Chinese Hamster ◽  
Human Cells ◽  
Dhfr Gene ◽  
Pyrimidine Dimers ◽  
Cross Links

My colleagues and I have discovered intragenomic heterogeneity in DNA repair in mammalian cells. Consequences of unrepaired DNA damage depend upon the precise location of the damage with respect to relevant genes. It is therefore important to understand rules governing accessibility of specific DNA sequences in chromatin to damage and repair. The efficiency of removal of pyrimidine dimers has been determined in the active dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. Repair within the gene was shown to be much more efficient than that in nontranscribed downstream sequences or in the genome overall. Preferential repair of active and essential genes such as DHFR may account for the fact that rodent cells are as uv-resistant as human cells in spite of their much lower overall repair efficiencies. In repair-proficient human cells the rate of repair in the DHFR gene is greater than that in the overall genome or in nontranscribed α-DNA sequences. The efficiency of removal of pyrimidine dimers is much higher in the transcribed than the nontranscribed DNA strands of the DHFR gene in both CHO and human cells. An excision–repair complex may be directly coupled to the transcription machinery to ensure early removal of transcription-blocking lesions in active genes. Sequences in the active c-abl proto-oncogene are repaired much more efficiently than are sequences containing the inactive c-mos proto-oncogene in Swiss mouse 3T3 cells. Tissue-specific and cell-specific differences in the coordinate regulation of proto-oncogene expression and DNA repair may account for corresponding differences in the carcinogenic response. Efficient replicative bypass of persisting psoralen monoadducts, but not interstrand cross-links, was demonstrated in the human DHFR gene. It is likely that most bulky lesions in mammalian DNA, other than cross-links, do not pose insurmountable problems for replication in vivo, but they must be removed from essential transcribed sequences to maintain cellular viability.Key words: DNA repair, chromatin, transcription, mammalian cells, pyrimidine dimers, ultraviolet light, DNA cross-links.

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

1998 ◽  
Vol 18 (6) ◽  
pp. 3563-3571 ◽  
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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