Nucleic Acids Research, 2003, Vol. 31, No. 12 3006-3015
© 2003 Oxford University Press
Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro
National Creative Research Initiative Center for Cell Cycle Control, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejoen, 305-701, Korea, 1 Department of Molecular Biology and Biochemistry, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, USA and 2 Department of Biology, Inha University, 3-Ga, Yonghyun-Dong, Nam-Ku, Inchon, 402-751, Korea
*To whom correspondence should be addressed. Tel: +82 42 869 2637; Fax: +82 42 869 8280; Email: yeonsooseo{at}mail.kaist.ac.kr
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.
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