Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1

Abstract

Upon DNA replication stress, stalled DNA replication forks serve as a platform to recruit many signaling proteins, leading to the activation of the DNA replication checkpoint. Activation of Rad53, a key effector kinase in the budding yeast Saccharomyces cerevisiae, is essential for stabilizing DNA replication forks during replication stress. Using an activity-based assay for Rad53, we found that Mrc1, a replication fork-associated protein, cooperates with Mec1 to activate Rad53 directly. Reconstitution of Rad53 activation using purified Mec1 and Mrc1 showed that the addition of Mrc1 stimulated a more than 70-fold increase in the ability of Mec1 to activate Rad53. Instead of increasing the catalytic activity of Mec1, Mrc1 was found to facilitate the phosphorylation of Rad53 by Mec1 via promotion of a stronger enzyme-substrate interaction between them. Further, the conserved C-terminal domain of Mrc1 was found to be required for Rad53 activation. These results thus provide insights into the role of the adaptor protein Mrc1 in activating Rad53 in the DNA replication checkpoint.Faithful replication of the genome is important for the survival of all organisms. During DNA replication, replication stress can arise from a variety of situations, including intrinsic errors made by DNA polymerases, difficulties in replicating repeated DNA sequences, and failures to repair damaged DNA caused by either endogenous oxidative agents or exogenous mutagens such as UV light and DNA-damaging chemicals (1-3). In eukaryotes, there is an evolutionarily conserved DNA replication checkpoint that becomes activated in response to DNA replication stress. It helps to stabilize DNA replication forks, block late replication origin firing, and delay mitosis and ultimately helps recovery from stalled replication forks after DNA repair (4 -7). Defects in the DNA replication checkpoint could result in elevated genomic instabilities, cancer development, or cell death (8, 9).Aside from replicating the genome, the DNA replication forks also provide a platform to assemble many signaling proteins that function in the DNA replication checkpoint. In the budding yeast Saccharomyces cerevisiae, Mec1, an ortholog of human ATR, 2 is a phosphoinositide 3-kinase-like kinase (PIKK) involved in sensing stalled DNA replication forks. Mec1 forms a protein complex with Ddc2 (ortholog of human ATRIP). The Mec1-Ddc2 complex is recruited to stalled replication forks through replication protein A (RPA)-coated single-stranded DNA (10, 11). The Mec3-Rad17-Ddc1 complex, a proliferating cell nuclear antigen (PCNA)-like checkpoint clamp and ortholog of the human 9-1-1 complex, was shown to be loaded onto the single-and double-stranded DNA junction of the stalled replication forks by the clamp loader Rad24-RFC complex (12). Once loaded, the Mec3-Rad17-Ddc1 complex stimulates Mec1 kinase activity (13). Dbp11 and its homolog TopBP1 in vertebrates are known components of the replication machinery (14). In addition to regulating the initiation of DNA replication, they were foun...

Mcm2-7 Is an Active Player in the DNA Replication Checkpoint Signaling Cascade via Proposed Modulation of Its DNA Gate

“…Previous results suggest that mediators in addition to Mrc1 (and possibly Csm3 and Tof1) are required in vivo to facilitate appropriate Rad53 activation. Under simplified conditions using either a purified biochemical system (73) or an engineered in vivo system (74), evidence indicates that the only limitation to Rad53 phosphorylation in the DRC is the physical association between Mec1 and Rad53. This physical connection is greatly stimulated by phosphorylated Mrc1, which binds to both kinases.…”

The Saccharomyces cerevisiae Rad6 Postreplication Repair and Siz1/Srs2 Homologous Recombination-Inhibiting Pathways Process DNA Damage That Arises in asf1 Mutants

“…All of the strains used were derived from the S288c strain RDKY3615 (MATa ura3-52 leu2⌬1 trp1⌬63 his3⌬200 lys2⌬Bgl hom3-10 ade2⌬1 ade8 hxt13::URA3) either by crossing with other RDKY3615 derivatives or by standard gene disruptions (7). The TAF tag added at the C terminus of PCNA has the structure His 6 -FLAG 3 -TEV cleavage site-(protein A) 2 , and the His 6 -FLAG 1 tag is a derivative of the TAF tag lacking the TEV site and the two protein A sequences (9,10,71). The detailed genotypes of the strains are listed in Table 1.…”

Extending the Interaction Repertoire of FHA and BRCT Domains

“…For instance, Dpb11 bridges the interaction between the 9-1-1 clamp and Mec1, leading to the full activation of the Mec1 kinase [24,25]. Mec1 has many roles at the stalled fork including facilitating the activation of the next downstream kinase in the pathway, Rad53 [26]. This is accomplished after Mec1 phosphorylates Mrc1-a protein naturally associated with the stalled replisome [27].…”