title: The Pol30-K196 residue plays a critical role in budding yeast DNA postreplication repair through interaction with Rad18.
abstract: PCNA plays critical roles in DNA replication and various DNA repair pathways including DNA damage tolerance (DDT). In budding yeast Saccharomyces cerevisiae, DDT (aka DNA postreplication repair, PRR) is achieved by sequential ubiquitination of PCNA encoded by POL30. Our previous studies revealed that two Arabidopsis PCNA genes were able to complement the essential function of POL30 in budding yeast, but failed to rescue the PRR activity. Here we hypothesize that a certain amino acid variation(s) is responsible for the difference, and identified K196 as a critical residue for the PRR activity. It was found that the pol30-K196V mutation abolishes Rad18 interaction and PRR activity, whereas nearby amino acid substitutions can partially restore Rad18 interaction and PRR activity. Together with the Pol30-Ub fusion data, we believe that we have identified a putative Rad18-binding pocket in Pol30 that is required for PCNA monoubiquitination and PRR.
authors.affiliation: College of Life Sciences, Capital Normal University, Beijing, 100048, China.
authors.forename: Li
authors.initials: L
authors.lastname: Fan
authors.affiliation: College of Life Sciences, Capital Normal University, Beijing, 100048, China; Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada. Electronic address: wei.xiao@usask.ca.
authors.forename: Wei
authors.initials: W
authors.lastname: Xiao
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004249:DNA Damage; D004260:DNA Repair; D004261:DNA Replication; D004271:DNA, Fungal / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques; D054875:Ubiquitination
chemical_list: D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C546594:POL30 protein, S cerevisiae; D018809:Proliferating Cell Nuclear Antigen; C063607:RAD18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008239:Lysine
keywords: DNA-damage tolerance; PCNA; Rad18 interaction; Ubiquitin