Multiple Rad5 Activities Mediate Sister Chromatid Recombination to Bypass DNA Damage at Stalled Replication Forks

DNA damage that blocks replication is bypassed in order to complete chromosome duplication and preserve cell viability and genome stability. Rad5, a PCNA polyubiquitin ligase and DNA-dependent ATPase in yeast, is orthologous to putative tumor suppressors and controls error-free damage bypass by an unknown mechanism. To identify the mechanism in vivo, we investigated the roles of Rad5 and analyzed the DNA structures that form during damage bypass at site-specific stalled forks present at replication origins. Rad5 mediated the formation of recombination-dependent, X-shaped DNA structures containing Holliday junctions between sister chromatids. Mutants lacking these damage-induced chromatid junctions were defective in resolving stalled forks, restarting replication, and completing chromosome duplication. Rad5 polyubiquitin ligase and ATPase domains both contributed to replication fork recombination. Our results indicate that multiple activities of Rad5 function coordinately with homologous recombination factors to enable replication template switch events that join sister chromatids at stalled forks and bypass DNA damage. [Display omitted] ► Rad5 is required to resolve forks stalled by DNA damage and to restart replication ► Homologous recombination factors function along with Rad5 in the same pathway ► Rad5 enables a Holliday junction to form between sister chromatids at a stalled fork ► Replication fork recombination requires Rad5 ATPase and ubiquitin ligase activities