PCNA ubiquitination is important,but not essential for translesion DNA synthesis in mammalian cells

Translesion dna synthesis (tls) is a dna damage tolerance mechanism in which specialized low-fidelity dna polymerases bypass replication-blocking lesions,and it is usually associated with mutagenesis. in saccharomyces cerevisiae a key event in tls is the monoubiquitination of pcna,which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. in mammals,however,there is a debate on the requirement for ubiquitinated pcna (pcna-ub) in tls. we show that uv-induced rpa foci,indicative of single-stranded dna (ssdna) regions caused by uv,accumulate faster and disappear more slowly in pcna k164r/k164r cells,which are resistant to pcna ubiquitination,compared to pcna +/+ cells,consistent with a tls defect. direct analysis of tls in these cells,using gapped plasmids with site-specific lesions,showed that tls is strongly reduced across uv lesions and the cisplatin-induced intrastrand gg crosslink. a similar effect was obtained in cells lacking rad18,the e3 ubiquitin ligase which monoubiquitinates pcna. consistently,cells lacking usp1,the enzyme that de-ubiquitinates pcna exhibited increased tls across a uv lesion and the cisplatin adduct. in contrast,cells lacking the rad5-homologs shprh and hltf,which polyubiquitinate pcna,exhibited normal tls. knocking down the expression of the tls genes rev3l,polh,or rev1 in pcna k164r/k164r mouse embryo fibroblasts caused each an increased sensitivity to uv radiation,indicating the existence of tls pathways that are independent of pcna-ub. taken together these results indicate that pcna-ub is required for maximal tls. however,tls polymerases can be recruited to damaged dna also in the absence of pcna-ub,and perform tls,albeit at a significantly lower efficiency and altered mutagenic specificity.