Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast

Abstract

The Schizosaccharomyces pombe pfh1+ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing. Mutations in the pfh1+ gene suppress a temperature-sensitive allele of cdc24+, which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing. In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27 or pol 3, all of which are involved in the Okazaki fragment metabolism. We show that a full-length Pfh1 fusion protein is active as a monomer. The helicase activity of Pfh1 displaced only short (<30 bp) duplex DNA regions efficiently in a highly distributive manner and was markedly stimulated by the presence of a replication-fork-like structure in the substrate. The temperature-sensitive phenotype of a dna2-C2 or a cdc24-M38 mutant was suppressed by pfh1-R20 (a cold-sensitive mutant allele of pfh1) and overexpression of wild-type pfh1+ abolished the ability of the pfh1 mutant alleles to suppress dna2-C2 and cdc24-M38. Purified Pfh1-R20 mutant protein displayed significantly reduced ATPase and helicase activities. These results indicate that the simultaneous loss-of-function mutations of pfh1+ and dna2+ (or cdc24+) are essential to restore the growth defect. Our genetic data indicate that the Pfh1 DNA helicase acts in concert with Cdc24 and Dna2 to process single-stranded DNA flaps generated in vivo by pol -mediated lagging strand displacement DNA synthesis.

Murine Pif1 Interacts with Telomerase and Is Dispensable for Telomere Function In Vivo

“…Although Scrrm3⌬ cells do not exhibit changes in GCR or mitochondrial genome stability, these cells do exhibit genetic instability, presumably due to replication fork pausing at specific chromosomal loci, such as the ribosomal DNA locus (31,32,45,59,68,69). ScRRM3 and ScPIF1 exhibit genetic interactions (including synthetic lethality) with many gene products required in DNA replication and S-phase checkpoint arrest (1,2,17,50,58,59,69,71). Thus, additional inactivation of other DNA helicases involved in DNA replication or repair or proteins essential for the S-phase checkpoint may yet reveal an interesting phenotype in mPif Ϫ/Ϫ animals.…”

“…ScPif1 and Rrm3 also play roles in the mitochondria, where they contribute to mitochondrial genome stability (21,37,51,52,67,70). Finally, both ScPif1 and Pfh1 interact genetically with the Dna2 helicase and cooperate to ensure the correct processing of Okazaki fragments during DNA replication (17,58).…”

Mapping genomic hotspots of DNA damage by a single-strand-DNA-compatible and strand-specific ChIP-seq method

“…As shown recently by two-dimensional gel analysis, in the nucleus, Pfh1 prevents replication fork stalling at hard-to-replicate sites, including RNA polymerase III (Pol III) transcribed tRNA and 5S rRNA genes (Sabouri et al 2012;Steinacher et al 2012). To catalog sites protected by Pfh1 in a genome-wide manner, we analyzed Rad52 distribution in two cold-sensitive mutants of pfh1, pfh1-R20 and pfh1-R23 (Tanaka et al 2002;Ryu et al 2004). In the R20 mutant, at the restrictive temperature of 20°C, dozens of Rad52 binding peaks were found throughout the genome, with the strongest ones near the centromeres of chromosomes 2 and 3 ( Fig.…”

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

“…The frequency in production of long flaps can be affected by the interactions of pol ␦/Dna2 with other proteins. For example, Pif1 helps create long flaps through its helicase activity in vitro (27) and in vivo (28), and the lethal phenotype of dna2⌬ was suppressed by pif1⌬ (28,29).…”