Upon DNA damage, cells activate signal transduction pathway which lead to cell cycle arrest and inhibition of DNA replication. Cell extracts from UV-irradiated HeLa cells, compared to those from non-irradiated cells, poorly support DNA replication, suggesting that damage-induced intra-S-phase arrest may be due to the activation of a regulatory mechanism rather than to the blockage of the replication machinery at the damaged DNA site. Previous studies showed that poor replication activity with extracts from UV-irradiated HeLa cells could be due to the lack of human replication protein A complex (RPA) in such extracts, since the addition of purified RPA reverses this inhibitory effect. In an attempt to address the molecular basis of this damage-induced inhibition of replication, we biochemically separated the RPA-containing fraction from the irradiated extracts and examined for RPA activity in replication. The RPA isolated from the irradiated cell extracts very poorly supported in vitro SV40 DNA replication with crude extracts lacking RPA or with purified replication proteins, suggesting that RPA from irradiated extracts is inactivated. The cytosolic extracts prepared from both DNA-dependent protein kinase (DNAPK) + and DNA-PK- ceils showed UV-induced inhibition of replication. However, its reversal by RPA was observed only with extracts from DNA-PK- cells, but not with extracts from DNA-PK+ cells, suggesting that DNA-PK is involved in DNA damage intra-S checkpoint control. A possible role of DNA-PK in this pathway will be discussed.
|Original language||English (US)|
|State||Published - Dec 1 1998|
ASJC Scopus subject areas
- Molecular Biology