20 nM

To maintain genomic integrity cells have to respond properly to a variety of exogenous and endogenous sources of DNA damage. DNA integrity is maintained by the coordinated action of DNA damage response mechanisms and DNA repair. In addition, there are also mechanisms of damage tolerance, such as translesion synthesis (TLS), which are important for survival after DNA damage but are potentially error-prone. Here, we investigate the role of DNA polymerase kappa (pol kappa) in TLS across alkylated lesions by silencing this polymerase (pol) in human cells using transient small RNA interference. We show that human pol kappa has a significant protective role against methyl nitrosourea (MNU)-associated cytotoxicity without affecting significantly mutagenicity. The increase in MNU-induced cytotoxicity when pol kappa is down-regulated was affected by the levels of O(6)-methylguanine DNA methyltransferase and fully abolished when mismatch repair (MMR) was defective. Following MNU treatment, the cell cycle profile was unaffected by the pol kappa status. The downregulation of pol kappa caused a severe delay in the onset of the second mitosis that was fully dependent on the presence of O(6)-methylguanine (O(6)-meGua) lesions. After MNU exposure, in the absence of pol kappa, the frequency of sister chromatid exchanges was unaffected whereas the induction of RAD 51 foci increased. We propose that pol kappa partially protects human cells from the MMR-dependent cytotoxicity of O(6)-meGua lesions by restoring the integrity of replicated duplexes containing single-stranded gaps generated opposite O(6)-meGua facilitated by RAD 51 binding.