Abstract
Base excision repair (BER) is highly conserved among various organisms as one of the major repair mechanism against altered bases in DNA. In single nucleotide BER pathway, a specific DNA glycosylase removes a damaged base. Resulting an apurinic/apyrimidinic site (AP site) is removed by DNA polymerase I, which fills the gap with a correct nucleotide to leave a nick to be sealed by DNA ligase III. X-ray repair cross complementing protein 1 (XRCC1) is known to interact physically with not only DNA ligase III but also other BER related proteins as a scaffold protein, suggesting that XRCC1 is involved in the whole BER pathway. To study the role of XRCC1 in the BER pathway against methyl methanesulfonate (MMS)-induced damages, we prepared XRCC1 knockdown cells with shRNA. We designed a shRNA against human XRCC1 (NCBI: NM 006297), and constructed the shRNA expression plasmid with psiRNA-hH1GFPzeo G2 (InvivoGen). We transfected the plasmids into HeLa RC355 cells with a liposome transfection agent, and selected the transfectant clones by zeocin resistance. With western blotting analysis, XRCC1 protein level in a knocodown (KD) clone was 44% of that in the negative control. Using colony-forming assay, we detected a significant decrease in survival of the KD clone at 1.5 mM MMS, as compared with the control. To reveal the effect of XRCC1 knockdown on methylpurine DNA glycosylase activity, ARP assay of the KD clone is in progress.
