To study the association between methylated endogenous DNA double-strand break and repair pathway

Abstract

DNA double-strand breaks (DSBs) are the type of DNA damage that is very harmful to cells. This event occurs when both of DNA strands are damaged. Spontaneous DSBs at the background level are called endogenous DSBs (EDSBs). From previous study, we found that EDSBs are generally hypermethylated and the hypermethylation is replication independent. EDSBs repair pathways are composed of at least three major pathways namely, ATM-dependent non-homologous end joining (NHEJ), DNA-PK-dependent NHEJ and homologous recombination (HR). NHEJ pathway is the cause of error-prone repair since the mechanism of this repair is the direct ligation of DNA ends. Both ATM and DNA-PK dependent NHEJ occur in G0 phase of the cell cycle. HR pathway is the major mechanism in S phase for error-free repair using an undamaged homologous sequence as a template for repair. Rad51, which catalyses the invasion of the broken ends of the DSB into the intact sister chromatid, is the key protein in this pathway. In this study, we aimed to identify which pathways are involved in the repair of hypermethylated EDSBs. We established siRNA technique to reduce the key proteins in each repair pathway, namely ATM, DNA-PKcs, Ku86 and Rad51. To measure the level and methylation of EDSBs in transfected cells, we performed L1-EDSB-LMPCR and COBRA-L1-EDSB, respectively. Stable transfection of DNA-PKcs siRNA in HeLa cells caused down-regulation not only of DNA-PKcs but also of ATM. EDSB methylation levels of DNA-PKcs siRNA cells are significantly lower than that of ATM siRNA transfected cells, especially in G0 phase, suggesting that the loss of DNA-PKcs compensated the influence of ATM deficiency on the methylation level of accumulated EDSBs. Thus, methylated EDSB is possibly repaired by ATM-dependent NHEJ, which is more precise than DNA-PK-dependent NHEJ. Additionally, there was no different in the level and methylation of EDSBs in Rad51 knock-down cells, indicating that hypermethylation of EDSBs does not depend on DNA replication. This study supports the notion that the increase of spontaneous mutation rate in genomic hypomethylation may be related to how differently the methylated and unmethylated EDSBs are processed.