Functional crosstalk between hOgg1 and the helicase domain of Cockayne syndrome group B protein.
We have previously reported that the Cockayne syndrome group B gene product (CSB) contributes to base excision repair (BER) of 8-hydroxyguanine (8-OH-Gua) and the importance of motifs V and VI of the putative helicase domains of CSB in BER of 8-OH-Gua. To further elucidate the function of CSB in BER, we investigated its role in the pathway involving human 8-OH-Gua glycosylase/apurinic lyase (hOgg1). Depletion of CSB protein with anti-CSB antibody reduced the 8-OH-Gua incision rate of wild type cell extracts but not of CSB null and motif VI mutant cell extracts, suggesting a direct contribution of CSB to the catalytic process of 8-OH-Gua incision and the importance of its motif VI in this pathway. Introduction of recombinant purified CSB partially complemented the depletion of CSB as shown by the recovery of the incision activity. This complementation could not fully recover the deficiency of the incision activity in WCE from CS-B null and mutant cell lines, suggesting that some additional factor(s) are necessary for the full activity. Electrophoretic mobility shift assays (EMSAs) showed a defect in binding of CSB null and motif VI mutant cell extracts to 8-OH-Gua-containing oligonucleotides. We detected less hOgg1 transcript and protein in the cell extracts from CS-B null and mutant cells, suggesting hOgg1 may be the missing component. Pull-down of hOgg1 by histidine-tagged CSB and co-localization of those two proteins after gamma-radiation indicated their co-existence in vivo, particularly under cellular stress. However, we did not detect any functional and physical interaction between purified CSB and hOgg1 by incision, gel shift and yeast two-hybrid assays, suggesting that even though hOgg1 and CSB might be in a common protein complex, they may not interact directly. We conclude that CSB functions in the catalysis of 8-OH-Gua BER and in the maintenance of efficient hOgg1 expression, and that motif VI of the putative helicase domain of CSB is crucial in these functions.
Tuo
Jingsheng
J
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Chen
Catheryne
C
Zeng
Xianmin
X
Christiansen
Mette
M
Bohr
Vilhelm A
VA
D020816:Amino Acid Motifs; D015153:Blotting, Western; D002457:Cell Extracts; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003057:Cockayne Syndrome / Q000235:genetics; D004247:DNA / Q000235:genetics*; D004265:DNA Helicases / Q000502:physiology*; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D045643:DNA Repair Enzymes; D043382:DNA-Formamidopyrimidine Glycosylase; D024202:Electrophoretic Mobility Shift Assay; D005455:Fluorescent Antibody Technique; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009699:N-Glycosyl Hydrolases / Q000502:physiology*; D000075223:Poly-ADP-Ribose Binding Proteins; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014162:Transfection; D020798:Two-Hybrid System Techniques
D002457:Cell Extracts; D017931:DNA Primers; D000075223:Poly-ADP-Ribose Binding Proteins; D011994:Recombinant Proteins; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; D004265:DNA Helicases; C078673:ERCC6 protein, human; D045643:DNA Repair Enzymes