Abstract
Pif1 is a multifunctional helicase and DNA processing enzyme that has roles in genome stability. The enzyme is conserved in eukaryotes and also found in some prokaryotes. The functions of human PIF1 (hPIF1) are also critical for survival of certain tumour cell lines during replication stress, making it an important target for cancer therapy. Crystal structures of hPIF1 presented here explore structural events along the chemical reaction coordinate of ATP hydrolysis at an unprecedented level of detail. The structures for the apo as well as the ground and transition states reveal conformational adjustments in defined protein segments that can trigger larger domain movements required for helicase action. Comparisons with the structures of yeast and bacterial Pif1 reveal a conserved ssDNA binding channel in hPIF1 that we show is critical for single-stranded DNA binding during unwinding, but not the binding of G quadruplex DNA. Mutational analysis suggests that while the ssDNA-binding channel is important for helicase activity, it is not used in DNA annealing. Structural differences, in particular in the DNA strand separation wedge region, highlight significant evolutionary divergence of the human PIF1 protein from bacterial and yeast orthologues.
Highlights
Accurate DNA replication requires a suite of enzymes including helicases that translocate on DNA
High resolution X-ray structures were determined for the helicase domain of human PIF1 (hPIF1) (Figure 1) corresponding to an idle cycle of ATP hydrolysis: structures of complexes with the ground state analogue AMP-PNP in two different crystal forms (1.13 A, 1.43 A ; Figure 1A), an apo structure complexed with three sulphate moieties determined at 1.44Aresolution (Figure 1B) and a low-resolution structure of a complex with the transition state analogue ADPAlF4−, Figure 1C
While the hPIF1-ADPAlF4− structure (Rfactor 17.9%; Rfree = 25.3%) mirrors a previously published low resolution (3.6 A ) crystal structure of the hPIF1 helicase domain (residues 200–641, pdb code: 5FHH, Rfactor 31.3%; Rfree = 35.5%, [26]), it is based on using the high-resolution structure of the hPIF1-AMP-PNP complex during molecular replacement, resulting in a more accurate model than the previously available structure (RMSCA = 2.0 A ; RMSoverall = 2.5 A )
Summary
Accurate DNA replication requires a suite of enzymes including helicases that translocate on DNA. Helicases can catalyse protein displacement from DNA [1] but they are known primarily for their ability to remodel DNA secondary structure [2] and generate single-stranded DNA (ssDNA) during DNA replication, repair, recombination or restart [3]. The purified yeast protein, ScPif, was shown to be a helicase [8] and nuclear DNA replication functions were identified [9]. Purified ScPif is a DNA-dependent ATPase and 5′–3′ helicase [8,18] that unwinds forked dsDNA substrates with ssDNA tails and RNA-DNA hybrids [19] and binds and unwinds G4 DNA [20]. Genome analysis has since identified at least one Pif1like gene in almost all eukaryotes and, curiously, some prokaryotes [23,24]
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