DOMAIN ANALYSIS OF M ACEIVORANS REPLICATION PROTEIN A 1

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The oligonucleotide/oligosaccharide-binding (OB) fold, a module ranging from 70-150 amino acids, is central to the architecture of single-stranded DNA-binding proteins. Single-stranded DNA-binding proteins are essential in diverse cellular processes. The bacterial single-stranded DNA-binding protein, a single polypeptide known as SSB, harbors a single OB fold. In contrast, the eukaryotic functional homolog, known as replication protein A (RPA), is a heterotrimeric protein containing multiple OB folds. In the methanogenic archaea, single polypeptide RPA proteins with multiple OB folds and a zinc finger domain have been described. The OB folds of these proteins were more similar to their eukaryotic counterparts than the bacterial ones. Here, we describe the functional analysis of a new form of RPA from the methanogenic archaeon Methanosarcina acetivorans. The novel RPA, designated MacRPA1, is comprised of four OB folds and lacks the zinc finger domain hitherto found in the RPA proteins of methanogens. MacRPA1 was compared with two other RPA proteins in M. acetivorans for their effects on DNA strand exchange, DNA synthesis, and cleavage of a flap by cognate proteins involved in these processes. MacRPA1 stimulated DNA strand exchange and primer extension reactions, however, it suppressed cleavage of a flap by the methanosarcinal homolog of flap endonuclease 1. N-terminal and C-terminal truncated derivatives of MacRPA1 were made and their properties were studied using biochemical and biophysical methods. Most interestingly, comparison of MacRPA1 with potential orthologs provided critical insight into a process that may serve to generate new OB folds in cells.