Abstract
The maintenance of genome integrity is regulated in part by chromatin structure and factors involved in the DNA damage response pathway. Nucleosome assembly is a highly regulated process that restores chromatin structure after DNA replication, DNA repair, and gene transcription. During S phase the histone chaperones Asf1, CAF-1, and Rtt106 coordinate to deposit newly synthesized histones H3-H4 onto replicated DNA in budding yeast. Here we describe synthetic genetic interactions between RTT106 and the DDC1-MEC3-RAD17 (9-1-1) complex, a sliding clamp functioning in the S phase DNA damage and replication checkpoint response, upon treatment with DNA damaging agents. The DNA damage sensitivity of rad17Δ rtt106Δ cells depends on the function of Rtt106 in nucleosome assembly. Epistasis analysis reveals that 9-1-1 complex components interact with multiple DNA replication-coupled nucleosome assembly factors, including Rtt106, CAF-1, and lysine residues of H3-H4. Furthermore, rad17Δ cells exhibit defects in the deposition of newly synthesized H3-H4 onto replicated DNA. Finally, deletion of RAD17 results in increased association of Asf1 with checkpoint kinase Rad53, which may lead to the observed reduction in Asf1-H3 interaction in rad17Δ mutant cells. In addition, we observed that the interaction between histone H3-H4 with histone chaperone CAF-1 or Rtt106 increases in cells lacking Rad17. These results support the idea that the 9-1-1 checkpoint protein regulates DNA replication-coupled nucleosome assembly in part through regulating histone-histone chaperone interactions.
Highlights
Genome stability is maintained in part by chromatin structure and checkpoint factors
These results indicate that the DNA damage sensitivity of double mutants containing the rtt106⌬ or a component of the 9-1-1 complex is due to defects other than cell cycle progression and initial signaling of the DNA damage checkpoint, supporting the idea that the CPT damage sensitivity observed in cells lacking Rtt[106] and components of 9-1-1 complex is most likely due to compromised nucleosome assembly in double mutant cells
We suggest that the observed increase in interactions between histone H3 and histone chaperone CAF-1 and Rtt[106] is due to free histones released from Asf1-H3-H4 complex in rad17⌬ mutant cells
Summary
Genome stability is maintained in part by chromatin structure and checkpoint factors. We describe synthetic genetic interactions between RTT106 and the DDC1-MEC3-RAD17 (9-1-1) complex, a sliding clamp functioning in the S phase DNA damage and replication checkpoint response, upon treatment with DNA damaging agents. It is proposed that the Rad53-Asf[1] interaction is regulated by Mec[1], which in turn regulates the function of Asf[1] and Hir[1], a histone chaperone involved in DNA replication-independent nucleosome assembly, in the maintenance of chromatin integrity (24). In an attempt to identify novel regulators of nucleosome assembly and the maintenance of genome integrity during DNA damage stress, we performed a synthetic genetic array analysis for factors that function in parallel to RTT106 in the response to the DNA damaging agent camptothecin (CPT), a topoisomerase I inhibitor. Our results suggest a novel role for the 9-1-1 complex in DNA replication-coupled nucleosome assembly
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