title: Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro.
abstract: We have previously shown that replication- protein A (RPA), the heterotrimeric single-stranded DNA binding protein of eukaryotes, plays a role in Okazaki fragment processing by acting as a molecular switch between the two endonucleases, Dna2 and Fen1, to ensure the complete removal of primer RNAs in Saccharomyces cerevisiae. The stimulation of Dna2 endonuclease activity by RPA requires direct protein-protein interaction. In this report we have analyzed genetically and biochemically the interaction of Dna2 with RPA. RFA1, the gene encoding the large subunit of RPA, displayed allele-specific interactions with DNA2 that included synthetic lethality and intergenic complementation. In addition, we identified physical and functional interactions between these proteins and found that RPA binds Dna2 predominantly through its large subunit, Rpa1. Consistent with the mapping of synthetic lethal mutations, robust interaction localizes to the C-termini of these proteins. Moreover, the N-terminal domains of Dna2 and Rpa1 appear to be important for a functional interaction because the N-terminal domain of RPA1 was required to maximally stimulate Dna2 endonuclease activity. We propose that a bimodal interaction of Dna2 with Rpa1 is important for Dna2 function both in vivo and in vitro. The relevance of each interaction with respect to the function of the Dna2 endonuclease activity is discussed.
authors.affiliation: National Creative Research Initiative Center for Cell Cycle Control, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejoen, 305-701, Korea.
authors.forename: Kwang-Hee
authors.initials: KH
authors.lastname: Bae
authors.forename: Hee-Sook
authors.initials: HS
authors.lastname: Kim
authors.forename: Sung-Ho
authors.initials: SH
authors.lastname: Bae
authors.forename: Ho-Young
authors.initials: HY
authors.lastname: Kang
authors.forename: Steven
authors.initials: S
authors.lastname: Brill
authors.forename: Yeon-Soo
authors.initials: YS
authors.lastname: Seo
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D000483:Alleles; D001665:Binding Sites; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D005804:Genes, Lethal; D009154:Mutation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D051716:Replication Protein A; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D021122:Protein Subunits; C082728:RFA1 protein, S cerevisiae; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004720:Endonucleases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae