In vitro analysis of UV-damage-induced inhibition of replication

We investigated DNA-damage-induced inhibition of replication by using an in vitro system, with which both replication and repair can be examined simultaneously. The system contains non-irradiated simian virus 40 (SV40) origin-containing DNA, UV-irradiated circular duplex DNA lacking an SV40 origin, and cell extracts that support both replication and repair activities. Using this system, we show that replication is significantly inhibited in the presence of UV-irradiated, but not non-irradiated, DNA and, to a lesser extent, repair activity is also inhibited by the presence of replication activity. In contrast, replication activity was not affected by UV-damaged DNA when the reactions were carried out with purified replication proteins, suggesting that protein factor(s) in the cell extracts are involved in the inhibition of replication that is triggered by DNA damage. Inhibition was efficiently reversed by the combined actions of proteins involved in both repair and replication, suggesting that the inhibition of replication observed in our system may be caused by the recruitment of replication proteins to damaged DNA sites.