Endonuclease V-mediated deoxyinosine excision repair in vitro.
Deoxyinosine (dI) in DNA can arise from hydrolytic or nitrosative deamination of deoxyadenosine. It is excised in a repair pathway that is initiated by endonuclease V, the nfi gene product, in Escherichia coli. Repair was studied in vitro using M13mp18 derived heteroduplexes containing a site-specific deoxyinosine. Unpaired dI/G mismatch resides within the recognition site for XhoI restriction endonucleases, permitting evaluation of repair occurring on deoxyinosine-containing DNA strand. Our results show that dI lesions were efficiently repaired in nfi(+)E. coli extracts but the repair level was much reduced in nfi mutant extracts. We subjected the deoxyinosine-containing heteroduplex to a purified system consisting of soluble endonuclease V fusion protein, DNA polymerase I, and DNA ligase, along with the four deoxynucleoside triphosphates. Interestingly we found these three proteins alone are sufficient to process the dI lesion efficiently. We also found that the 3'-exonuclease activity of DNA polymerase I is sufficient to remove the dI lesion in this minimum reconstituted assay.
Lee
Chia-Chia
CC
Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei 100-63, Taiwan, ROC.
Yang
Ya-Chien
YC
Goodman
Steven D
SD
Yu
Yung-Hsu
YH
Lin
Shwu-Bin
SB
Kao
Jau-Tsuen
JT
Tsai
Keh-Sung
KS
Fang
Woei-Horng
WH
D011088:DNA Ligases / Q000378:metabolism; D053843:DNA Mismatch Repair; D004256:DNA Polymerase I / Q000378:metabolism; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D003641:Deamination; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007288:Inosine / Q000031:analogs & derivatives* / Q000378:metabolism
D004269:DNA, Bacterial; D007288:Inosine; D004256:DNA Polymerase I; D043224:Deoxyribonuclease (Pyrimidine Dimer); D011088:DNA Ligases; C012271:deoxyinosine