In situ analysis of repair processes for oxidative DNA damage in mammalian cells.
Oxidative DNA damage causes blocks and errors in transcription and replication, leading to cell death and genomic instability. Although repair mechanisms of the damage have been extensively analyzed in vitro, the actual in vivo repair processes remain largely unknown. Here, by irradiation with an UVA laser through a microscope lens, we have conditionally produced single-strand breaks and oxidative base damage at restricted nuclear regions of mammalian cells. We showed, in real time after irradiation by using antibodies and GFP-tagged proteins, rapid and ordered DNA repair processes of oxidative DNA damage in human cells. Furthermore, we characterized repair pathways by using repair-defective mammalian cells and found that DNA polymerase beta accumulated at single-strand breaks and oxidative base damage by means of its 31- and 8-kDa domains, respectively, and that XRCC1 is essential for both polymerase beta-dependent and proliferating cell nuclear antigen-dependent repair pathways of single-strand breaks. Thus, the repair of oxidative DNA damage is based on temporal and functional interactions among various proteins operating at the site of DNA damage in living cells.
Lan
Li
L
Department of Molecular Genetics, Institute of Development, Aging, and Cancer, Tohoku University, Seiryomachi 4-1, Sendai 980-8575, Japan.
Nakajima
Satoshi
S
Oohata
Yoshitsugu
Y
Takao
Masashi
M
Okano
Satoshi
S
Masutani
Mitsuko
M
Wilson
Samuel H
SH
Yasui
Akira
A
D001483:Base Sequence; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D010084:Oxidation-Reduction; D034622:RNA Interference / Q000502:physiology; D017382:Reactive Oxygen Species; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000076105:X-ray Repair Cross Complementing Protein 1
D017931:DNA Primers; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D017382:Reactive Oxygen Species; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D019951:DNA Polymerase beta