Replicative DNA helicases expose the two strands of the double helix to the 
replication apparatus, but accessory helicases are often needed to help forks 
move past naturally occurring hard-to-replicate sites, such as tightly bound 
proteins, RNA/DNA hybrids, and DNA secondary structures. Although the 
Schizosaccharomyces pombe 5'-to-3' DNA helicase Pfh1 is known to promote fork 
progression, its genomic targets, dynamics, and mechanisms of action are largely 
unknown. Here we address these questions by integrating genome-wide 
identification of Pfh1 binding sites, comprehensive analysis of the effects of 
Pfh1 depletion on replication and DNA damage, and proteomic analysis of Pfh1 
interaction partners by immunoaffinity purification mass spectrometry. Of the 
621 high confidence Pfh1-binding sites in wild type cells, about 40% were sites 
of fork slowing (as marked by high DNA polymerase occupancy) and/or DNA damage 
(as marked by high levels of phosphorylated H2A). The replication and integrity 
of tRNA and 5S rRNA genes, highly transcribed RNA polymerase II genes, and 
nucleosome depleted regions were particularly Pfh1-dependent. The association of 
Pfh1 with genomic integrity at highly transcribed genes was S phase dependent, 
and thus unlikely to be an artifact of high transcription rates. Although Pfh1 
affected replication and suppressed DNA damage at discrete sites throughout the 
genome, Pfh1 and the replicative DNA polymerase bound to similar extents to both 
Pfh1-dependent and independent sites, suggesting that Pfh1 is proximal to the 
replication machinery during S phase. Consistent with this interpretation, Pfh1 
co-purified with many key replisome components, including the hexameric MCM 
helicase, replicative DNA polymerases, RPA, and the processivity clamp PCNA in 
an S phase dependent manner. Thus, we conclude that Pfh1 is an accessory DNA 
helicase that interacts with the replisome and promotes replication and 
suppresses DNA damage at hard-to-replicate sites. These data provide insight 
into mechanisms by which this evolutionarily conserved helicase helps preserve 
genome integrity.