title: Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.
abstract: Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms.
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58108, USA.
authors.forename: Padmaja L
authors.initials: PL
authors.lastname: Ghospurkar
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58108, USA.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Wilson
authors.affiliation: Department of Oral Biology, University of Nebraska Medical Center, Lincoln, NE 68583, USA.
authors.forename: Shengqin
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58108, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Herauf
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58108, USA.
authors.forename: Jenna
authors.initials: J
authors.lastname: Steffes
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58108, USA.
authors.forename: Erica N
authors.initials: EN
authors.lastname: Mueller
authors.affiliation: Department of Oral Biology, University of Nebraska Medical Center, Lincoln, NE 68583, USA.
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Oakley
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58108, USA; Interdisciplinary Cellular and Molecular Biology Program, North Dakota State University, Fargo, ND 58108, USA. Electronic address: stuart.haring@ndsu.edu.
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Haring
mesh_terms: D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D004261:DNA Replication; D006367:HeLa Cells; D006801:Humans; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D012333:RNA, Messenger; D051716:Replication Protein A; C516794:RPA2 protein, human
keywords: DNA damage response; N-terminus; Phosphorylation; RPA