The yeast Pif1p DNA helicase preferentially unwinds RNA DNA substrates.
Pif1p is the prototypical member of the PIF1 family of DNA helicases, a subfamily of SFI helicases conserved from yeast to humans. Baker's yeast Pif1p is involved in the maintenance of mitochondrial, ribosomal and telomeric DNA and may also have a general role in chromosomal replication by affecting Okazaki fragment maturation. Here we investigate the substrate preferences for Pif1p. The enzyme was preferentially active on RNA-DNA hybrids, as seen by faster unwinding rates on RNA-DNA hybrids compared to DNA-DNA hybrids. When using forked substrates, which have been shown previously to stimulate the enzyme, Pif1p demonstrated a preference for RNA-DNA hybrids. This preferential unwinding could not be correlated to preferential binding of Pif1p to the substrates that were the most readily unwound. Although the addition of the single-strand DNA-binding protein replication protein A (RPA) stimulated the helicase reaction on all substrates, it did not diminish the preference of Pif1p for RNA-DNA substrates. Thus, forked RNA-DNA substrates are the favored substrates for Pif1p in vitro. We discuss these findings in terms of the known biological roles of the enzyme.
Boulé
Jean-Baptiste
JB
Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. jboule@princeton.edu
Zakian
Virginia A
VA
D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D051716:Replication Protein A / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012965:Sodium Chloride / Q000737:chemistry; D013379:Substrate Specificity
D009841:Oligonucleotides; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D012965:Sodium Chloride; D012313:RNA; D004247:DNA; C085463:PIF1 protein, S cerevisiae; D004265:DNA Helicases