Rad9, a 53BP1 Ortholog of Budding Yeast, Is Insensitive to Spo11-Induced Double-Strand Breaks During Meiosis

Exogenous double-strand breaks (DSBs) induce a DNA damage response during mitosis as well as meiosis. The DNA damage response is mediated by a cascade involving Mec1/Tel1 (ATR/ATM) and Rad53 (Chk2) kinases. Meiotic cells are programmed to form DSBs for the initiation of meiotic recombination. In budding yeast, Spo11-mediated meiotic DSBs activate Mec1/Tel1, but not Rad53; however, the mechanism underlying the insensitivity of Rad53 to meiotic DSBs remains largely unknown. In this study, we found that meiotic cells activate Rad53 in response to exogenous DSBs and that this activation is dependent on an epigenetic marker, Dot1-dependent histone H3K79 methylation, which becomes a scaffold of an Rad53 mediator, Rad9, an ortholog of 53BP1. In contrast, Rad9 is insensitive to meiotic programmed DSBs. This insensitiveness of Rad9 derives from its inability to bind to the DSBs. Indeed, artificial tethering of Rad9 to the meiotic DSBs activated Rad53. The artificial activation of Rad53 kinase in meiosis decreases the repair of meiotic DSBs. These results suggest that the suppression of Rad53 activation is a key event in initiating a meiotic program that repairs programmed DSBs.

Exceptional origin activation revealed by comparative analysis in two laboratory yeast strains

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two common laboratory strains of Saccharomyces cerevisiae challenged by hydroxyurea (HU), an inhibitor of the ribonucleotide reductase. By doing so we gained understanding of the impact on origin activation by three factors: replication checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Our analysis recapitulated the previous finding that the majority of origins are subject to checkpoint control by the Rad53 kinase when cells were treated with HU. In addition, origins either subject to Rad53 checkpoint control or impervious to it are largely concordant between the two strains. However, these two strains also produced different dynamics of origin activation. First, the W303-RAD53 cells showed a significant reduction of fork progression than A364a-RAD53 cells. This phenotype was accompanied by an elevated level of Rad53 phosphorylation in W303-RAD53 cells. Second, W303-rad53K227A checkpoint-deficient cells activated a greater number of origins accompanied by global reduction of ssDNA across all origins compared to A364a-rad53K227A cells; and this is correlated with lower expression level of the mutant protein in W303 than in A364a. We also show that sequence polymorphism in the consensus motifs of the replication origins plays a minor role in determining origin usage. Remarkably, eight strain-specific origins lack the canonical 11-bp consensus motif for autonomously replicating sequences in either strain background. Finally, we identified a new class of origins that are only active in checkpoint-proficient cells, which we named “Rad53-dependent origins”. The only discernible feature of these origins is that they tend to overlap with an open reading frame, suggesting previously unexplored connection between transcription and origin activation. Our study presents a comprehensive list of origin usage in two diverse yeast genetic backgrounds, fine-tunes the different categories of origins with respect to checkpoint control, and provokes further exploration of the interplay between origin activation and transcription.Author SummaryComparative analysis of origins of replication in two laboratory yeast strains reveals new insights into origin activation, regulation and dependency on the Rad53 checkpoint kinase.

Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase

Across eukaryotes, checkpoints maintain the order of cell cycle events in the face of DNA damage or incomplete replication. Although a wide array of DNA lesions activates the checkpoint kinases, whether and how this response differs in different phases of the cell cycle remains poorly understood. The S-phase checkpoint for example results in the slowing of replication, which in the budding yeast Saccharomyces cerevisiae is caused by Rad53 kinase-dependent inhibition of the initiation factors Sld3 and Dbf4. Despite this, we show here that Rad53 phosphorylates both of these substrates throughout the cell cycle at the same sites as in S-phase, suggesting roles for this pathway beyond S-phase. Indeed we show that Rad53-dependent inhibition of Sld3 and Dbf4 limits re-replication in G2/M phase, preventing inappropriate gene amplification events. In addition we show that inhibition of Sld3 and Dbf4 after DNA damage in G1 phase prevents premature replication initiation at all origins at the G1/S transition. This study redefines the scope and specificity of the ‘S-phase checkpoint’ with implications for understanding the roles of this checkpoint in the majority of cancers that lack proper cell cycle controls.

Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal extension (NTE) of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and −6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.

Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

ABSTRACTEukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal tail of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, but requires Rad53 activation by trans-autophosphorylation, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition.

Sae2 promotes DNA damage resistance by removing the Mre11–Rad50–Xrs2 complex from DNA and attenuating Rad53 signaling

The Mre11–Rad50–Xrs2/NBS1 (MRX/N) nuclease/ATPase complex plays structural and catalytic roles in the repair of DNA double-strand breaks (DSBs) and is the DNA damage sensor for Tel1/ATM kinase activation. Saccharomyces cerevisiae Sae2 can function with MRX to initiate 5′-3′ end resection and also plays an important role in attenuation of DNA damage signaling. Here we describe a class of mre11 alleles that suppresses the DNA damage sensitivity of sae2Δ cells by accelerating turnover of Mre11 at DNA ends, shutting off the DNA damage checkpoint and allowing cell cycle progression. The mre11 alleles do not suppress the end resection or hairpin-opening defects of the sae2Δ mutant, indicating that these functions of Sae2 are not responsible for DNA damage resistance. The purified MP110LRX complex shows reduced binding to single- and double-stranded DNA in vitro relative to wild-type MRX, consistent with the increased turnover of Mre11 from damaged sites in vivo. Furthermore, overproduction of Mre11 causes DNA damage sensitivity only in the absence of Sae2. Together, these data suggest that it is the failure to remove Mre11 from DNA ends and attenuate Rad53 kinase signaling that causes hypersensitivity of sae2Δ cells to clastogens.

A novel method to stabilize weak protein complexes for crystallographic studies

Most protein interactions mediating critical steps in cellular pathways are transient and, hence, are difficult to capture using structural approaches. An example from Saccharomyces cerevisiae is the interaction between checkpoint effector Rad53 kinase and replication initiation Dbf4-Cdc7 kinase. Dbf4 is the regulatory and Cdc7 the catalytic subunit of this kinase, which functions in activating replication origins. When a replication fork is damaged, the checkpoint response activates Rad53, which binds transiently, yet specifically, to Dbf4 and this, in turn, inhibits the activity of Dbf4-Cdc7. The outcome of this interaction prevents activation of late origins during replication stress. Our laboratory has extensively characterized the Dbf4-Rad53 interaction, thereby providing an excellent system to probe new ways to stabilize a weak protein complex. The N-terminal forkhead associated (FHA) domain of Rad53 mediates the interaction with the BRCA-1 C-terminus (BRCT) domain of Dbf4. FHA and BRCT domains are modular domains, in which their N- and C-termini lie on the same face. Thus, we decided to stabilize the Dbf4-Rad53 complex using linkers of different lengths to join the two proteins. We generated four different Dbf4-linker-Rad53 fusions and characterized them biochemically, as well as structurally using small angle X-ray scattering. Only one of the fusions yielded crystals suitable for crystallographic analysis, and we solved the structure by molecular replacement. The four copies of the fusion in the asymmetric unit showed identical Dbf4-Rad53 interfaces that were in agreement with previous studies characterizing the Dbf4-Rad53 interaction. Importantly, the interaction in the crystal structure occurs inter- rather than intra-molecularly suggesting that the linker increases local protein concentration but does not impose complex formation. In a broader sense, our work reveals general trends that can be used to design linkers to capture weak protein complexes.