title: Characterization of the interaction between Rfa1 and Rad24 in Saccharomyces cerevisiae.
abstract: Maintaining the integrity of the genome requires the high fidelity duplication of the genome and the ability of the cell to recognize and repair DNA lesions. The heterotrimeric single stranded DNA (ssDNA) binding complex Replication Protein A (RPA) is central to multiple DNA processes, which are coordinated by RPA through its ssDNA binding function and through multiple protein-protein interactions. Many RPA interacting proteins have been reported through large genetic and physical screens; however, the number of interactions that have been further characterized is limited. To gain a better understanding of how RPA functions in DNA replication, repair, and cell cycle regulation and to identify other potential functions of RPA, a yeast two hybrid screen was performed using the yeast 70 kDa subunit, Replication Factor A1 (Rfa1), as a bait protein. Analysis of 136 interaction candidates resulted in the identification of 37 potential interacting partners, including the cell cycle regulatory protein and DNA damage clamp loader Rad24. The Rfa1-Rad24 interaction is not dependent on ssDNA binding. However, this interaction appears affected by DNA damage. The regions of both Rfa1 and Rad24 important for this interaction were identified, and the region of Rad24 identified is distinct from the region reported to be important for its interaction with Rfc2 5. This suggests that Rad24-Rfc2-5 (Rad24-RFC) recruitment to DNA damage substrates by RPA occurs, at least partially, through an interaction between the N terminus of Rfa1 and the C terminus of Rad24. The predicted structure and location of the Rad24 C-terminus is consistent with a model in which RPA interacts with a damage substrate, loads Rad24-RFC at the 5' junction, and then releases the Rad24-RFC complex to allow for proper loading and function of the DNA damage clamp.
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58108, United States of America.
authors.forename: Gunjan
authors.initials: G
authors.lastname: Piya
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58108, United States of America.
authors.forename: Erica N
authors.initials: EN
authors.lastname: Mueller
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58108, United States of America.
authors.forename: Heather K
authors.initials: HK
authors.lastname: Haas
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58108, United States of America.
authors.forename: Padmaja L
authors.initials: PL
authors.lastname: Ghospurkar
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58108, United States of America.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Wilson
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58108, United States of America.
authors.forename: Jaime L
authors.initials: JL
authors.lastname: Jensen
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58108, United States of America; Interdisciplinary Program in Cellular and Molecular Biology, North Dakota State University, Fargo, ND, 58108, United States of America.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Colbert
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND, 58108, United States of America; Interdisciplinary Program in Cellular and Molecular Biology, North Dakota State University, Fargo, ND, 58108, United States of America.
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Haring
mesh_terms: D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051716:Replication Protein A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D020798:Two-Hybrid System Techniques
chemical_list: D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; C088385:RAD24 protein, S cerevisiae; C082728:RFA1 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D008741:Methyl Methanesulfonate