Cut5 Is Required for the Binding of Atr and DNA Polymerase α to Genotoxin-damaged Chromatin

Abstract

DNA damage triggers the assembly of checkpoint signaling proteins on chromatin that activate the Chk1 signaling pathway and block S-phase progression. Here we show that genotoxin-induced Chk1 activation requires Cut5 (Mus101/TopBP1) in a process that is independent of the role of Cut5 in DNA replication. Analysis of the role of Cut5 in checkpoint activation revealed that it associated with chromatin following DNA damage in a process that required RPA. Additionally, Cut5 was required for the recruitment of Atr, DNA polymerase ␣, and Rad1 but not RPA to chromatin following DNA damage. Taken together, these results demonstrate that Cut5 plays an integral role in the recruitment and assembly of the Chk1 signaling cascade components following DNA damage.Checkpoint signaling pathways maintain genomic integrity by blocking cell cycle progression, influencing DNA repair, and in the event of severe DNA damage, prompting programmed cell death (1-3). Central to these signaling pathways are the phosphatidylinositol 3-kinase-related kinases (PIKK) 1 Atm (ataxia-telangiectasia mutated) and Atr . Atm along with its downstream transducing partner Chk2 responds to DNA double-strand breaks (2). In contrast, during S-phase, other types of DNA damage independently recruit Atr, claspin, and the heterotrimeric Rad9-Hus1-Rad1 (9-1-1) complex to chromatin (4 -7), where they mediate activation of the protein kinase Chk1 (5,8,9). Activated Chk1 then stabilizes stalled replication forks and impedes S-phase progression by blocking the firing of origins of replication (10 -12).Although much progress has been made in the identification and ordering of checkpoint signaling proteins, it is clear that additional components also participate in the signaling pathways. Possible candidate participants in the pathways are the Cut5/Dpb11/Mus101 orthologs. Cut5 and Dpb11 were identified in Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively (13,14). Although these proteins are required for DNA replication, they are also essential for activation of the replication checkpoint, indicating that they participate in checkpoint signaling. In higher eukaryotes, orthologs of Cut5 and Dpb11 have also been identified. Drosophila Mus101, human TopBP1, and Xenopus Cut5 (also known as Mus101) are highly related to one another and more distantly related to Cut5/Dpb11 (15-18). In Drosophila, hypomorphic alleles of the Mus101 gene confer sensitivity to genotoxins (19). Likewise, human TopBP1 forms nuclear foci that co-localize with 53BP1, BRCA1, and NBS in response to replication arrest and ionizing radiation (20,21). In addition, human TopBP1 is phosphorylated by PIKKs in response to genotoxic stress and interacts with the checkpoint signaling proteins Rad9 and 53BP1 (20,21). These observations suggest that the putative TopBP1/ Mus101/Cut5 orthologs found in higher eukaryotes also participate cellular responses to DNA damage; however, the role of Cut5 orthologs in checkpoint signaling has not been explored.Besides their role in checkpoint activatio...

The Rad9–Hus1–Rad1 (9–1–1) clamp activates checkpoint signaling via TopBP1

“…Rad9 was previously reported to interact with TopBP1 (Makiniemi et al 2001;St. Onge et al 2003), a checkpoint protein that plays a role in Chk1 activation (Parrilla-Castellar and Karnitz 2003). Intriguingly, TopBP1 also contains eight BRCA 1 C-terminal (BRCT) domains ( Fig.…”

TopBP1 and ATR Colocalization at Meiotic Chromosomes: Role of TopBP1/Cut5 in the Meiotic Recombination Checkpoint

“…Both TopBP1 protein homologues in yeast (S. pombe Rad4/Cut5 and S. cerevisiae Dpb11) are required for DNA replication and for the DNA damage and DNA replication checkpoints and act upstream of effector kinases spChk1, spCds1, and scRad53 (Saka and Yanagida, 1993;Araki et al, 1995;McFarlane et al, 1997;Wang and Elledge, 1999;Harris et al, 2003). Recently, studies in Xenopus indicate that the TopBP1 homologue, xCut5, is required for ATR binding to the sites of DNA damage (Parrilla-Castellar and Karnitz, 2003).…”

Replication Protein A and the Mre11·Rad50·Nbs1 Complex Co-localize and Interact at Sites of Stalled Replication Forks

“…Further evidence that RPA acts as a common intermediate for signaling stalled replication forks/DNA damage is demonstrated by the RPA-dependent binding of Cut5 to chromatin after DNA damage and its subsequent recruitment of ATR and DNA polymerase ␣ to chromatin (15). In that report, Parrilla-Castellar and Karnitz (15) put forth a model suggesting that ssDNA generated at a stalled replication fork is coated with RPA, leading to Cut5 chromatin association. Cut5 then facilitates the chromatin association of ATR and DNA Pol␣, which in turn leads to the loading of the 9-1-1 complex (15).…”

“…RPA is also required to recruit and activate Rad17 complexes for checkpoint signaling in vivo (14). Further evidence that RPA acts as a common intermediate for signaling stalled replication forks/DNA damage is demonstrated by the RPA-dependent binding of Cut5 to chromatin after DNA damage and its subsequent recruitment of ATR and DNA polymerase ␣ to chromatin (15). In that report, Parrilla-Castellar and Karnitz (15) put forth a model suggesting that ssDNA generated at a stalled replication fork is coated with RPA, leading to Cut5 chromatin association.…”

“…In that report, Parrilla-Castellar and Karnitz (15) put forth a model suggesting that ssDNA generated at a stalled replication fork is coated with RPA, leading to Cut5 chromatin association. Cut5 then facilitates the chromatin association of ATR and DNA Pol␣, which in turn leads to the loading of the 9-1-1 complex (15). These data have given credence to the hypothesis that RPA-coated ssDNA acts as a common intermediate for signaling-stalled replication forks and/or DNA damage and subsequent recruitment and activation of DNA damage response proteins.…”