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Molecular and Cellular Biology, December 1998, p. 7225-7234, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification and Characterization of the Fourth Single-Stranded-DNA Binding Domain of Replication Protein A

Steven J. Brill* and Suzanne Bastin-Shanower

Department of Molecular Biology, Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08855

Received 28 April 1998/Returned for modification 28 May 1998/Accepted 9 September 1998

Replication protein A (RPA), the heterotrimeric single-stranded-DNA (ssDNA) binding protein (SSB) of eukaryotes, contains two homologous ssDNA binding domains (A and B) in its largest subunit, RPA1, and a third domain in its second-largest subunit, RPA2. Here we report that Saccharomyces cerevisiae RPA1 contains a previously undetected ssDNA binding domain (domain C) lying in tandem with domains A and B. The carboxy-terminal portion of domain C shows sequence similarity to domains A and B and to the region of RPA2 that binds ssDNA (domain D). The aromatic residues in domains A and B that are known to stack with the ssDNA bases are conserved in domain C, and as in domain A, one of these is required for viability in yeast. Interestingly, the amino-terminal portion of domain C contains a putative Cys4-type zinc-binding motif similar to that of another prokaryotic SSB, T4 gp32. We demonstrate that the ssDNA binding activity of domain C is uniquely sensitive to cysteine modification but that, as with gp32, ssDNA binding is not strictly dependent on zinc. The RPA heterotrimer is thus composed of at least four ssDNA binding domains and exhibits features of both bacterial and phage SSBs.

* Corresponding author. Mailing address: Department of Molecular Biology, Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Rutgers University, Piscataway, NJ 08855. Phone: (732) 235-4197. Fax: (732) 235-4880. E-mail: brill{at}mbcl.rutgers.edu.

Molecular and Cellular Biology, December 1998, p. 7225-7234, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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