RNase H2 of Saccharomyces cerevisiae is a complex of three proteins

Abstract

The composition of RNase H2 has been a long-standing problem. Whereas bacterial and archaeal RNases H2 are active as single polypeptides, the Saccharomyces cerevisiae homolog, Rnh2Ap, when expressed in Escherichia coli, fails to produce an active RNase H2. By affinity chromatography purification and identification of polypeptides associated with a tagged S.cerevisiae Rnh2Ap, we obtained a complex of three proteins [Rnh2Ap (Rnh201p), Ydr279p (Rnh202p) and Ylr154p (Rnh203p)] that together are necessary and sufficient for RNase H2 activity [correction]. Deletion of the gene encoding any one of the proteins or mutations in the catalytic site in Rnh2A led to loss of RNase H2 activity. Even when S.cerevisiae RNase H2 is catalytically compromised, it still exhibits a preference for cleavage of the phosphodiester bond on the 5' side of a ribonucleotide-deoxyribonucleotide sequence in substrates mimicking RNA-primed Okazaki fragments or a single ribonucleotide embedded in a duplex DNA. Interestingly, Ydr279p and Ylr154p have homologous proteins only in closely related species. The multisubunit nature of S.cerevisiae RNase H2 may be important both for structural purposes and to provide a means of interacting with other proteins involved in DNA replication/repair and transcription.

Roles of SGS1, MUS81, and RAD51 in the repair of lagging-strand replication defects in Saccharomyces cerevisiae

“…One of the genes we isolated was RNH202, which encodes one of the subunits of RNase H2 (Jeong et al 2004). …”

“…Most species have two classes of RNases H, RNase HI/H1 and RNase HII/H2 (Ohtani et al 1999), and both enzymes specifically target the RNA portion of RNA-DNA/DNA or RNA/ DNA duplex (Ohtani et al 1999;Qiu et al 1999;Jeong et al 2004). Whereas bacterial RNases H2 are active as single polypeptides (Itaya 1990;Chapados et al 2001), RNase H2 of yeast consists of a heterotrimeric protein complex encoded by RNH201,RNH202, and RNH203 (Jeong et al 2004).…”

“…Whereas bacterial RNases H2 are active as single polypeptides (Itaya 1990;Chapados et al 2001), RNase H2 of yeast consists of a heterotrimeric protein complex encoded by RNH201,RNH202, and RNH203 (Jeong et al 2004). Rnh202 and Rnh203 are required in this complex to activate the catalytic activity of Rnh201 (Jeong et al 2004).…”

“…Whereas bacterial RNases H2 are active as single polypeptides (Itaya 1990;Chapados et al 2001), RNase H2 of yeast consists of a heterotrimeric protein complex encoded by RNH201,RNH202, and RNH203 (Jeong et al 2004). Rnh202 and Rnh203 are required in this complex to activate the catalytic activity of Rnh201 (Jeong et al 2004). Although RNase H1 is important for the development of higher cells (Filippov et al 2001;Cerritelli et al 2003), loss of both RNases H in singlecelled eukaryotes does not result in severe growth defects (Ray and Hines 1995;Arudchandran et al 2000).…”

Mouse models for Aicardi–Goutières syndrome provide clues to the molecular pathogenesis of systemic autoimmunity

“…The A subunit carries the catalytic centre while the B and C subunits provide scaffolding and sites for protein interactions [23,41]. All three subunits of the yeast and human enzyme are required for activity in vitro [40,42] and AGS can be caused by mutations in any of the three genes [40]. The mutations found in AGS patients seem to reduce but not to abrogate activity of the enzyme, and bi-allelic null mutations in any of the three Rnaseh2 genes have not been described in humans.…”