DNA replication through hard-to-replicate sites, including both highly transcribed RNA Pol II and Pol III genes, requires the S. pombe Pfh1 helicase

Abstract

Replication forks encounter impediments as they move through the genome, including natural barriers due to stable protein complexes and highly transcribed genes. Unlike lesions generated by exogenous damage, natural barriers are encountered in every S phase. Like humans, Schizosaccharomyces pombe encodes a single Pif1 family DNA helicase, Pfh1. Here, we show that Pfh1 is required for efficient fork movement in the ribosomal DNA, the mating type locus, tRNA, 5S ribosomal RNA genes, and genes that are highly transcribed by RNA polymerase II. In addition, converged replication forks accumulated at all of these sites in the absence of Pfh1. The effects of Pfh1 on DNA replication are likely direct, as it had high binding to sites whose replication was impaired in its absence. Replication in the absence of Pfh1 resulted in DNA damage specifically at those sites that bound high levels of Pfh1 in wild-type cells and whose replication was slowed in its absence. Cells depleted of Pfh1 were inviable if they also lacked the human TIMELESS homolog Swi1, a replisome component that stabilizes stalled forks. Thus, Pfh1 promotes DNA replication and separation of converged replication forks and suppresses DNA damage at hard-to-replicate sites.

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

“…S5). Thus, DNA lesion hotspots in pfh1-R20 are intimately related to Pol III genes, consistent with the known role of Pfh1 in overcoming replication block at Pol III genes (Sabouri et al 2012;Steinacher et al 2012).…”

“…3D). We suspect this correlation is related to the observations that in Pfh1-depleted cells tRNA genes became polar fork barriers that caused stronger elevation of replication pausing and recombination when replication and transcription move in opposite directions (Sabouri et al 2012;Steinacher et al 2012). …”

“…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).…”

Determination of the biochemical properties of full-length human PIF1 ATPase

“…18 Pfh1p also participates in lagging strand DNA maturation and DNA replication through hard-to-replicate sites. 20,21 Due to difficulties in the purification…”

Sequential eviction of crowded nucleoprotein complexes by the exonuclease RecBCD molecular motor

“…Nucleoprotein complexes are the primary source of replication fork stalling (46), and their presence represents a major challenge to genome integrity (1,37,38,(47)(48)(49). Indeed, prokaryotic and eukaryotic replisomes both require accessory helicases to clear tightly bound proteins from DNA (25,(50)(51)(52)(53)(54). For instance, the E. coli replisome requires the accessory helicases Rep and UvrD to prevent replication fork collapse on encountering RNAP and other types of high-affinity nucleoprotein complexes (25,46).…”