Effect of the cell cycle on translesion DNA synthesis

One of the main functions of TLS is to bypass DNA lesions that escaped repair, or were formed during replication, and may cause replication blocks. It was therefore assumed that TLS occurs exclusively in the S phase of the cell cycle. We analyzed the cell cycle-dependence of TLS by fractionating cell populations enriched for specific stages of the cell cycle using centrifugal elutriation, and analyzing their ability to perform TLS. We found that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. Using immune-staining with anti-RPA antibodies we showed that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persisted into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase  and Rev1. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions lags behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. This was not merely the result of the presence of gaps in DNA, since the cellular composition of the TLS machinery changed during the cell cycle, and became up-regulated in S and G2. The uncoupling of TLS from chromosomal replication may function to maintain efficient replication, so it can progress despite the presence of DNA lesions, with TLS lagging behind, and completing patching of any gaps left behind.