Rfc5, in Cooperation with Rad24, Controls DNA Damage Checkpoints throughout the Cell Cycle in Saccharomyces cerevisiae

Abstract

RAD24 and RFC5 are required for DNA damage checkpoint control in the budding yeast Saccharomyces cerevisiae. Rad24 is structurally related to replication factor C (RFC) subunits and associates with RFC subunits Rfc2, Rfc3, Rfc4, and Rfc5. rad24⌬ mutants are defective in all the G 1 -, S-, and G 2 /M-phase DNA damage checkpoints, whereas the rfc5-1 mutant is impaired only in the S-phase DNA damage checkpoint. Both the RFC subunits and Rad24 contain a consensus sequence for nucleoside triphosphate (NTP) binding. To determine whether the NTP-binding motif is important for Rad24 function, we mutated the conserved lysine 115 residue in this motif. The rad24-K115E mutation, which changes lysine to glutamate, confers a complete loss-of-function phenotype, while the rad24-K115R mutation, which changes lysine to arginine, shows no apparent phenotype. Although neither rfc5-1 nor rad24-K115R single mutants are defective in the G 1 -and G 2 /M-phase DNA damage checkpoints, rfc5-1 rad24-K115R double mutants become defective in these checkpoints. Coimmunoprecipitation experiments revealed that Rad24 K115R fails to interact with the RFC proteins in rfc5-1 mutants. Together, these results indicate that RFC5, like RAD24, functions in all the G 1 -, S-and G 2 /M-phase DNA damage checkpoints and suggest that the interaction of Rad24 with the RFC proteins is essential for DNA damage checkpoint control.Eukaryotic cells employ a set of surveillance mechanisms to coordinate cell cycle events by permitting the onset of one event only after the completion of the preceding event. The mechanisms that ensure the proper ordering of cell cycle events have been termed checkpoint controls (10). DNA damage triggers the activation of checkpoint pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. Other checkpoints are activated when DNA replication is blocked. Failure to respond properly to DNA alterations may result in genomic instability, a mutagenic condition that predisposes organisms to cancer (5, 24).The cell cycle is transiently arrested at different stages depending on the phase at which DNA damage occurs. Three responses have been characterized in the budding yeast Saccharomyces cerevisiae, known as the G 1 -, S-and G 2 /M-phase DNA damage checkpoints (16). Genetic studies have identified genes that are involved in all three checkpoints. These include RAD9, RAD17, RAD24, MEC3, DDC1, MEC1(ESR1), and RAD53 (SPK1 or MEC2) (1,17,18,22,23,(30)(31)(32)(33)(43)(44)(45). Several lines of genetic evidence have suggested that RAD17, RAD24, MEC3, and DDC1 operate in the same checkpoint pathway, while RAD9 functions separately (17,18,20). Indeed, Ddc1, Mec3, and Rad17 physically interact with each other, suggesting that they function as a complex (13). RAD53 encodes a dual-specificity protein kinase (35), and Mec1 belongs to the ATM protein family (12,28). Rad53 is phosphorylated in response to DNA damage in a MEC1-dependent manner (26, 39). DNA damage-induced Rad53 phosphorylation is also dependent on R...

Requirement for ATP by the DNA Damage Checkpoint Clamp Loader

“…The rad24-K115E mutation results in a complete loss of function phenotype (15). A K55R mutation in the RFC4 gene displays a mild checkpoint defect, whereas the K55E mutant shows no phenotype (14).…”

“…In contrast, the ATP-binding domain in Rfc1 is dispensable for clamp loading in vitro and rfc1-K359E mutants (with a mutation in the conserved lysine of the Walker A motif) show virtually no defects in vivo (13,14). However, the analogous lysine 3 glutamic acid mutation in the RAD24 gene results in a complete loss of function phenotype for the DNA damage checkpoint (15). We have determined that ATP binding to Rad24 is essential for complex formation with the Rad17/3/1 clamp and the observed in vivo that defect can be directly interpreted as a failure to load the clamp.…”

“…Plasmid pBL766-E (2 M ori URA3 GST-rad24-K115E) containing rad24-K115E was made by swapping a restriction fragment containing the mutation from plasmid YlpT-Rad24-K115E (a gift of Dr. Ken Sugimoto) into pBL766 (15). Plasmids pBL422-4R (2 M ori URA3 RFC2 RFC3 rfc4-K55R RFC5) and pBL422-4E (2 M ori URA3 RFC2 RFC3 rfc4-K55E RFC5) were made by swapping in appropriate restriction fragments from pBL410-R and pBL410-E, respectively (13,14).…”

Smc5–Smc6 complex suppresses gross chromosomal rearrangements mediated by break-induced replications

“…The smc6-9 strains carrying an additional mutation in sensors of the RAD24 branch DNA damage checkpoint such as rad24, rad17, or ddc1 [44], all increased the GCR rates synergistically compared to strains carrying each mutation (Table 5). An additional smc6-9 mutation in the rfc5-1 strain, which is defective in the DNA replication checkpoint [45,46] showed GCR formation rate comparable to the one caused by the rfc5-1 single mutation ( Table 5). The replication defects of rfc5-1 could be suppressed by multicopy PCNA expression [47].…”

Colocalization of human Rad17 and PCNA in late S phase of the cell cycle upon replication block

“…In budding yeast the interaction of the hRad17 homologue Rad24 with the small RF-C subunits is essential for DNA damage checkpoint control throughout the cell cycle (Naiki et al, 2000). Furthermore, Rad24 interacts functionally with the small RF-C subunit RF-C5 in the DNA replication and in the DNA damage checkpoint pathways.…”