Rfc4 Interacts with Rpa1 and Is Required for Both DNA Replication and DNA Damage Checkpoints in Saccharomyces cerevisiae

Abstract

The large subunit of replication protein A (Rpa1) consists of three single-stranded DNA binding domains and an N-terminal domain (Rpa1N) of unknown function. To determine the essential role of this domain we searched for mutations that require wild-type Rpa1N for viability in yeast. A mutation in RFC4, encoding a small subunit of replication factor C (RFC), was found to display allele-specific interactions with mutations in the gene encoding Rpa1 (RFA1). Mutations that map to Rpa1N and confer sensitivity to the DNA synthesis inhibitor hydroxyurea, such as rfa1-t11, are lethal in combination with rfc4-2. The rfc4-2 mutant itself is sensitive to hydroxyurea, and like rfc2 and rfc5 strains, it exhibits defects in the DNA replication block and intra-S checkpoints. RFC4 and the DNA damage checkpoint gene RAD24 were found to be epistatic with respect to DNA damage sensitivity. We show that the rfc4-2 mutant is defective in the G 1 /S DNA damage checkpoint response and that both the rfc4-2 and rfa1-t11 strains are defective in the G 2 /M DNA damage checkpoint. Thus, in addition to its essential role as part of the clamp loader in DNA replication, Rfc4 plays a role as a sensor in multiple DNA checkpoint pathways. Our results suggest that a physical interaction between Rfc4 and Rpa1N is required for both roles.Replication protein A (RPA) is a highly conserved eukaryotic single-stranded DNA (ssDNA) binding protein that was originally identified as a human factor required for simian virus 40 (SV40) DNA replication in vitro (19,84,86). As the eukaryotic equivalent of the Escherichia coli ssDNA binding protein (Ecssb), RPA plays multiple roles in DNA metabolism, including DNA replication, repair, and recombination (85). In addition to participating in both the initiation and elongation of DNA replication, RPA is required for nucleotide excision repair in vitro and physically interacts with XPA, XPG, and XPF (30,39,62,69). RPA is also required for genetic recombination and physically interacts with Rad52 and other factors (1,22,47,75).RPA is a heterotrimeric complex. In the budding yeast Saccharomyces cerevisiae it is composed of the Rpa1 (69 kDa), Rpa2 (36 kDa), and Rpa3 (13 kDa) subunits, which are encoded by RFA1, RFA2, and RFA3, respectively. Each subunit is essential for viability in yeast, and all three subunits are required for SV40 DNA replication in vitro (12,18,25,31,32). Rpa1 exhibits strong ssDNA binding activity on its own, and a subcomplex of Rpa2 and Rpa3 binds ssDNA weakly (7, 85). We have shown that yeast Rpa1 consists of four functional domains: an 18-kDa N-terminal domain that lacks ssDNA binding activity (Rpa1N) and three tandem ssDNA binding domains (SBDs; see Fig. 1A) (11, 56). SBD-A and SBD-B are structurally homologous as determined by X-ray crystallographic analysis of human RPA (hRPA), while the C-terminal domain (SBD-C) contains a C4-type zinc-finger motif (6,8,11). These three ssDNA binding domains share amino acid sequence similarity with each other and with the central region of Rpa2 (11). All fo...

Onset of the DNA Replication Checkpoint in the Early Drosophila Embryo

“…In Drosophila it is known that dRPA2 exists in two different phosphorylation states: unphosphorylated during late anaphase to telophase and phosphorylated from midprophase to early anaphase (Mitsis 1995). In yeast, Xenopus extracts, and human cells it has been shown that the RPA complex regulates Chk1 via ataxia-telangiectasia Rad3-related kinase (ATR) (Longhese et al 1996;Kim and Brill 2001;Costanzo et al 2003;Zou and Elledge 2003). ATR can initiate the phosphorylation of Chk1, which leads to a cell-cycle arrest (Liu et al 2000).…”

G2 damage checkpoints: what is the turn-on?

“…Although these effects could be indirect and related to a failure to undergo DNA replication, one study has shown that RPA is needed for ATR to be recruited to ssDNA generated in a replication-independent manner (Costanzo et al, 2003). In addition, a mutation, rfa1-t11, in the large subunit of yeast RPA that allows DNA replication prevents full activation of the checkpoint and promotes more rapid adaptation following DNA damage (Kim and Brill, 2001;Pellicioli et al, 2001). This mutant also fails to recruit Ddc2 (ATRIP) to ssDNA in vivo .…”

Unwind and slow down: checkpoint activation by helicase and polymerase uncoupling: Figure 1.

“…9-1-1 complex loading by Rad17 also requires RPA-ssDNA but is independent of ATR-ATRIP (Ellison and Stillman 2003;. Furthermore, RPA mutants that abolish interactions between RPA and replication factor C (RFC) in vitro cause checkpoint deficiencies in vivo (Kim and Brill 2001). Thus, there may be multiple receptors for the RPA-ssDNA ligand, and the original problem of how diverse DNA structures could activate a common signaling molecule seems to have been turned on its head.…”

“…Second, depletion of RPA from human cells with RNA interference prevents ATR-ATRIP localization to nuclear foci and phosphorylation of the ATR substrate Chk1 in response to UV radiation . Third, mutants in yeast RPA exhibit a checkpoint defect (Longhese et al 1996;Kim and Brill 2001). Fourth, ATRIP can bind to RPA-ssDNA .…”