Multi-BRCT Domain Protein Brc1 Links Rhp18/Rad18 and γH2A To Maintain Genome Stability during S Phase

ABSTRACT

DNA replication involves the inherent risk of genome instability, since replisomes invariably encounter DNA lesions or other structures that stall or collapse replication forks during the S phase. In the fission yeast Schizosaccharomyces pombe, the multi-BRCT domain protein Brc1, which is related to budding yeast Rtt107 and mammalian PTIP, plays an important role in maintaining genome integrity and cell viability when cells experience replication stress. The C-terminal pair of BRCT domains in Brc1 were previously shown to bind phosphohistone H2A (γH2A) formed by Rad3/ATR checkpoint kinase at DNA lesions; however, the putative scaffold interactions involving the N-terminal BRCT domains 1 to 4 of Brc1 have remained obscure. Here, we show that these domains bind Rhp18/Rad18, which is an E3 ubiquitin protein ligase that has crucial functions in postreplication repair. A missense allele in BRCT domain 4 of Brc1 disrupts binding to Rhp18 and causes sensitivity to replication stress. Brc1 binding to Rhp18 and γH2A are required for the Brc1 overexpression suppression of smc6-74, a mutation that impairs the Smc5/6 structural maintenance of chromosomes complex required for chromosome integrity and repair of collapsed replication forks. From these findings, we propose that Brc1 provides scaffolding functions linking γH2A, Rhp18, and Smc5/6 complex at damaged replication forks.

KEYWORDS:

• Brc1
• Rhp18
• Rad18
• BRCT domain
• DNA damage
• DNA repair
• DNA replication stress
• genome integrity

ACKNOWLEDGMENTS

We thank Matthew O'Connell for plasmids and technical insights, Oliver Limbo for technical support, and members of the Russell laboratory and Nick Boddy for helpful discussions.

This research was supported by NIH grants GM059347, CA077325, and CA117638 to P.R.

We declare no conflicts of interest.