Studies on the base excision repair (BER) complex in Pyrococcus furiosus

2009 ◽  
Vol 37 (1) ◽  
pp. 79-82 ◽  
Author(s):  
Shinichi Kiyonari ◽  
Saki Tahara ◽  
Maiko Uchimura ◽  
Tsuyoshi Shirai ◽  
Sonoko Ishino ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Pyrococcus Furiosus ◽  
Excision Repair ◽  
Repair Process ◽  
Nuclear Antigen ◽  
Physical Interaction ◽  
Ap Endonuclease ◽  
Reading Frame ◽  
Base Excision

We have been studying the functions of PCNA (proliferating-cell nuclear antigen) for the assembly and reassembly of the replisome during replication fork progression. We have identified the functional interactions between PCNA and several proteins involved in DNA replication and repair from Pyrococcus furiosus. We recently reported that the activity of UDG (uracil–DNA glycosylase) in P. furiosus (PfuUDG) is stimulated by PCNA (PfuPCNA) in vitro, and identified an atypical PCNA-binding site, AKTLF, in the PfuUDG protein. To understand further the function of the complex in the BER (base excision repair) process, we investigated the AP (apurinic/apyrimidinic) endonuclease, which can process the BER pathway after uracil removal by UDG. Interestingly, one candidate ORF (open reading frame) for the AP endonuclease was found in the operon containing the gene encoding UDG in the P. furiosus genome. However, this ORF did not exhibit any activity. Instead, we identified the AP endonuclease activity from the other candidate gene products, and designated the protein as PfuAP. We discovered a physical interaction between PfuAP and PfuPCNA, suggesting the formation of a BER complex in one of the repair systems in P. furiosus.

scholarly journals Spontaneous Mutagenesis Is Enhanced in Apex Heterozygous Mice

2004 ◽  
Vol 24 (18) ◽  
pp. 8145-8153 ◽  
Author(s):  
Jessica Huamani ◽  
C. Alex McMahan ◽  
Damon C. Herbert ◽  
Robert Reddick ◽  
John R. McCarrey ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Germ Line ◽  
Excision Repair ◽  
Spontaneous Mutant ◽  
Spermatogenic Cells ◽  
Ap Endonuclease ◽  
Repair Activity ◽  
Base Excision ◽  
Mutant Frequency

ABSTRACT Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex +/−) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex +/− lacI + mice compared to frequencies from Apex +/+ lacI + littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex +/− lacI + mice were significantly elevated twofold compared to levels for 9-month-old Apex +/+ lacI + control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.

In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts

1999 ◽  
pp. 301-315 ◽  
Author(s):  
Guido Frosina ◽  
Enrico Cappelli ◽  
Paola Fortini ◽  
Eugenia Dogliotti
Keyword(s):  
Mammalian Cell ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Cell Extracts ◽  
Base Excision

scholarly journals A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb1 and DNA polymerase β

2010 ◽  
Vol 39 (8) ◽  
pp. 3156-3165 ◽  
Author(s):  
De-Sheng Pei ◽  
Xiao-Jie Yang ◽  
Wei Liu ◽  
Jeroen E. J. Guikema ◽  
Carol E. Schrader ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Dna Polymerase Β ◽  
Ap Endonuclease ◽  
Regulatory Circuit ◽  
Ap Endonuclease 1 ◽  
Base Excision

scholarly journals MSH2–MSH6 stimulates DNA polymerase η, suggesting a role for A:T mutations in antibody genes

2005 ◽  
Vol 201 (4) ◽  
pp. 637-645 ◽  
Author(s):  
Teresa M. Wilson ◽  
Alexandra Vaisman ◽  
Stella A. Martomo ◽  
Patsa Sullivan ◽  
Li Lan ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Base Excision Repair ◽  
Somatic Hypermutation ◽  
Excision Repair ◽  
Cytidine Deaminase ◽  
Abasic Site ◽  
Cell Extracts ◽  
Activation Induced Cytidine Deaminase ◽  
Base Excision

Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2–MSH6 mismatch repair heterodimer and DNA polymerase (pol) η, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2–MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol η in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2–MSH6 stimulates the catalytic activity of pol η in vitro. These observations suggest that the interaction between MSH2–MSH6 and DNA pol η stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs.

A new approach utilizing real-time qPCR to detect in vitro base excision repair

DNA Repair ◽  
2010 ◽  
Vol 9 (8) ◽  
pp. 898-906 ◽  
Author(s):  
Honghai Zhang ◽  
Yunjin Zang ◽  
Yu Sun ◽  
Ronghua Jin ◽  
Hao Wu ◽  
...  
Keyword(s):  
Real Time ◽  
Base Excision Repair ◽  
Excision Repair ◽  
New Approach ◽  
Base Excision ◽  
Real Time Qpcr

scholarly journals DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 3005-3014 ◽  
Author(s):  
Nivedita P. Khairnar ◽  
Hari S. Misra
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Base Excision Repair ◽  
Deinococcus Radiodurans ◽  
Excision Repair ◽  
Strand Break ◽  
Klenow Fragment ◽  
E Coli ◽  
Base Excision

The Deinococcus radiodurans R1 genome encodes an X-family DNA repair polymerase homologous to eukaryotic DNA polymerase β. The recombinant deinococcal polymerase X (PolX) purified from transgenic Escherichia coli showed deoxynucleotidyltransferase activity. Unlike the Klenow fragment of E. coli, this enzyme showed short patch DNA synthesis activity on heteropolymeric DNA substrate. The recombinant enzyme showed 5′-deoxyribose phosphate (5′-dRP) lyase activity and base excision repair function in vitro, with the help of externally supplied glycosylase and AP endonuclease functions. A polX disruption mutant of D. radiodurans expressing 5′-dRP lyase and a truncated polymerase domain was comparatively less sensitive to γ-radiation than a polX deletion mutant. Both mutants showed higher sensitivity to hydrogen peroxide. Excision repair mutants of E. coli expressing this polymerase showed functional complementation of UV sensitivity. These results suggest the involvement of deinococcal polymerase X in DNA-damage tolerance of D. radiodurans, possibly by contributing to DNA double-strand break repair and base excision repair.

AP endonuclease and poly(ADP-ribose) polymerase-1 interact with the same base excision repair intermediate

DNA Repair ◽  
2004 ◽  
Vol 3 (6) ◽  
pp. 581-591 ◽  
Author(s):  
Cheryl Cistulli ◽  
Olga I Lavrik ◽  
Rajendra Prasad ◽  
Esther Hou ◽  
Samuel H Wilson
Keyword(s):  
Base Excision Repair ◽  
Excision Repair ◽  
Ap Endonuclease ◽  
Base Excision
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