title: The DNA damage response and checkpoint adaptation in Saccharomyces cerevisiae: distinct roles for the replication protein A2 (Rfa2) N-terminus.
abstract: In response to DNA damage, two general but fundamental processes occur in the cell: (1) a DNA lesion is recognized and repaired, and (2) concomitantly, the cell halts the cell cycle to provide a window of opportunity for repair to occur. An essential factor for a proper DNA-damage response is the heterotrimeric protein complex Replication Protein A (RPA). Of particular interest is hyperphosphorylation of the 32-kDa subunit, called RPA2, on its serine/threonine-rich amino (N) terminus following DNA damage in human cells. The unstructured N-terminus is often referred to as the phosphorylation domain and is conserved among eukaryotic RPA2 subunits, including Rfa2 in Saccharomyces cerevisiae. An aspartic acid/alanine-scanning and genetic interaction approach was utilized to delineate the importance of this domain in budding yeast. It was determined that the Rfa2 N-terminus is important for a proper DNA-damage response in yeast, although its phosphorylation is not required. Subregions of the Rfa2 N-terminus important for the DNA-damage response were also identified. Finally, an Rfa2 N-terminal hyperphosphorylation-mimetic mutant behaves similarly to another Rfa1 mutant (rfa1-t11) with respect to genetic interactions, DNA-damage sensitivity, and checkpoint adaptation. Our data indicate that post-translational modification of the Rfa2 N-terminus is not required for cells to deal with "repairable" DNA damage; however, post-translational modification of this domain might influence whether cells proceed into M-phase in the continued presence of unrepaired DNA lesions as a "last-resort" mechanism for cell survival.
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota 58108.
authors.forename: Padmaja L
authors.initials: PL
authors.lastname: Ghospurkar
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota 58108.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Wilson
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota 58108.
authors.forename: Amber L
authors.initials: AL
authors.lastname: Severson
authors.affiliation: Department of Biological Sciences, Cedar Crest College, Allentown, Pennsylvania 18104.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Klein
authors.affiliation: Department of Biological Sciences, Cedar Crest College, Allentown, Pennsylvania 18104.
authors.forename: Sakina K
authors.initials: SK
authors.lastname: Khaku
authors.affiliation: Department of Biological Sciences, Cedar Crest College, Allentown, Pennsylvania 18104.
authors.forename: André P
authors.initials: AP
authors.lastname: Walther
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota 58108 Interdisciplinary Cellular and Molecular Biology Program, North Dakota State University, Fargo, North Dakota 58108 stuart.haring@ndsu.edu.
authors.forename: Stuart J
authors.identifier: http://orcid.org/0000-0002-6169-3547
authors.initials: SJ
authors.lastname: Haring
mesh_terms: D059447:Cell Cycle Checkpoints; D004249:DNA Damage; D004260:DNA Repair; D004271:DNA, Fungal / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051716:Replication Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004271:DNA, Fungal; C082727:RFA2 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins
keywords: DNA damage; Replication Protein A; checkpoint adaptation; genetic interaction; phosphorylation