title: The Rad5 helicase activity is dispensable for error-free DNA post-replication repair.
abstract: DNA post-replication repair (PRR) functions to bypass replication-blocking lesions and is subdivided into two parallel pathways: error-prone translesion DNA synthesis and error-free PRR. While both pathways are dependent on the ubiquitination of PCNA, error-free PRR utilizes noncanonical K63-linked polyubiquitinated PCNA to signal lesion bypass through template switch, a process thought to be dependent on Mms2-Ubc13 and a RING finger motif of the Rad5 ubiquitin ligase. Previous in vitro studies demonstrated the ability of Rad5 to promote replication fork regression, a function dependent on its helicase activity. To investigate the genetic and mechanistic relationship between fork regression in vitro and template switch in vivo, we created and characterized site-specific mutations defective in the Rad5 RING or helicase activity. Our results indicate that both the Rad5 ubiquitin ligase and the helicase activities are exclusively involved in the same error-free PRR pathway. Surprisingly, the Rad5 helicase mutation abolishes its physical interaction with Ubc13 and the K63-linked PCNA polyubiquitin chain assembly. Indeed, physical fusions of Rad5 with Ubc13 bypass the requirement for either the helicase or the RING finger domain. Since the helicase domain overlaps with the SWI/SNF chromatin-remodelling domain, our findings suggest a structural role of this domain and that the Rad5 helicase activity is dispensable for error-free lesion bypass.
authors.affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Lindsay G
authors.initials: LG
authors.lastname: Ball
authors.affiliation: College of Life Sciences, Capital Normal University, Beijing 100048, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
authors.affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Susan
authors.initials: S
authors.lastname: Blackwell
authors.affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Michelle D
authors.initials: MD
authors.lastname: Hanna
authors.affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada.
authors.forename: Amanda D
authors.initials: AD
authors.lastname: Lambrecht
authors.affiliation: College of Life Sciences, Capital Normal University, Beijing 100048, China; Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada. Electronic address: wei.xiao@usask.ca.
authors.forename: Wei
authors.initials: W
authors.lastname: Xiao
mesh_terms: D001665:Binding Sites; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics*; D017354:Point Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D004271:DNA, Fungal; D018809:Proliferating Cell Nuclear Antigen; D029701:Saccharomyces cerevisiae Proteins; C471610:UBC13 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; C076225:RAD5 protein, S cerevisiae; D004265:DNA Helicases
keywords: Helicase; Post-replication repair; RING finger; Replication fork regression; Saccharomyces cerevisiae; Template switch