Roles of Replication Protein A and DNA-dependent Protein Kinase in the Regulation of DNA Replication following DNA Damage

Abstract

Exposure of mammalian cells to DNA damage-inducing agents (DDIA) inhibits ongoing DNA replication. The molecular mechanism of this inhibition remains to be elucidated. We employed a simian virus 40 (SV40) based in vitro DNA replication assay to study biochemical aspects of this inhibition. We report here that the reduced DNA replication activity in extracts of DDIA-treated cells is partly caused by a reduction in the amount of replication protein A (RPA). We also report that the dominant inhibitory effect is caused by the DNAdependent protein kinase (DNA-PK) which inactivates SV40 T antigen (TAg) by phosphorylation. The results demonstrate that RPA and DNA-PK are involved in the regulation of viral DNA replication after DNA damage and suggest that analogous processes regulate cellular DNA replication with the DNA-PK targeting the functional homologues of TAg.Inhibition of DNA replication in eukaryotic cells is one of the earliest effects of radiation to be reported. Camptothecin (CPT) 1 and other DNA damage-inducing agents (DDIA) exert similar inhibitory effects on DNA replication in actively growing cells. It has been documented that a major component of this inhibition derives from a delay in the initiation of unreplicated origins (1, 2). Although this response was first observed several decades ago, the underlying molecular mechanisms remain largely unknown but ATM is thought to play an important role. More recently, evidence has been presented suggesting that the inhibition observed in DNA replication in response to DDIA is equivalent to the activation of a checkpoint in the S-phase and that it requires in yeast the products of MEC1 and RAD53 genes, homologues of ATM and Chk2,. It is important to establish the mechanisms of regulation of DNA replication after DNA damage and the biochemical functions of the above genes in the process. We investigated the regulation of DNA replication after DNA damage using a simian virus 40 (SV40) based in vitro DNA replication assay, and have shown that extracts of cells exposed to DDIA have reduced activity for in vitro DNA replication and inhibit, in a dominant fashion, the ability of extracts from nontreated cells to promote in vitro DNA replication (7,8).In the SV40-based DNA replication assay, replication of plasmids containing the SV40 origin of DNA (ori ϩ DNA) replication is accomplished in vitro with either crude cytoplasmic extracts or proteins purified from such extracts with SV40 T antigen (TAg) as the only noncellular protein (9 -13). It is believed that all cellular proteins required in this assay function in a manner similar to that in vivo (10 -14). We have reported that the degree of inhibition of DNA replication in cells exposed either to x-rays or CPT is comparable with that measured in vitro using extracts prepared from cells exposed to these agents (7,8). We subsequently focused on the molecular characterization of this inhibition. In the present study, we provide evidence that following DNA damage two processes contribute to the inhibition of in vitro ...

Adozelesin Triggers DNA Damage Response Pathways and Arrests SV40 DNA Replication through Replication Protein A Inactivation

“…The presence of transinhibitors makes it difficult to evaluate whether RPA has also been inactivated in extracts from cells treated with bizelesin, camptothecin, or gamma radiation. One recent report indicates that treatment of cells with camptothecin both induces a transacting inhibitor (which appears to be the DNA-dependent protein kinase acting on SV40 T antigen) and results in a 50% decrease in levels of RPA (26). However, this report gave no indication that camptothecin treatment resulted in RPA inactivation.…”

“…The rescue of SV40 DNA replication by the addition of RPA is reminiscent of results previously seen with extracts from UV-treated or hyperthermic cells (23,44). However, addition of RPA to extracts from camptothecin-treated cell extracts cannot rescue SV40 DNA replication (25,26). Similarly, and consistent with our results showing that bizelesin treatment induces a trans-acting inhibitor of DNA replication (11), addition of RPA cannot rescue SV40 DNA replication activity in bizelesin-treated cell extracts (data not shown).…”

“…However, following treatment of cells by the anti-cancer drug camptothecin or by gamma radiation, a trans-dominant negative factor was found to be present in treated cell extracts (24,25). Addition of purified RPA to these cell extracts could not rescue in vitro SV40 DNA replication activity (25,26). The different effects on SV40 DNA replication and the differing nature of the DNA-damaging agents have made it difficult to draw general conclusions about the mechanisms of DNA replication arrest following DNA damage.…”

Replication protein A is an independent prognostic indicator with potential therapeutic implications in colon cancer

“…22 It has been reported that tirapazamine (TPZ), a hypoxia-selective cytotoxin which is currently being examined in phase II and III clinical trials, greatly inhibits DNA synthesis and thus cell proliferation, through direct damage to or intracellular redistribution of RPA protein. [28][29][30][31][32] Using an in vitro DNA replication assay, Peters et al 22 observed that extracts from TPZ-treated colon cancer cells had a reduced ability to support in vitro DNA replication which was accompanied by a reduction in the levels of RPA. Addition of recombinant RPA restored replication activity to control levels.…”

DNA damage checkpoint control in cells exposed to ionizing radiation

“…First indications for a checkpoint in S phase (also termed intra-S phase checkpoint) came from the observation that exposure of cells to ionizing radiation causes a transient inhibition in the incorporation of radioactive precursors into nascent DNA (Painter, 1986;Lavin and Schroeder, 1988;Larner et al, 1997). Although the recognition that this effect reflects an active cellular response initiated by DNA damage is relatively recent (Lamb et al, 1989;Cleaver et al, 1990;Wang et al, 1995Wang et al, , 1999, early work allowed the detailed characterization of its salient cellular manifestations.…”