The evidence for conservation and possible functions of G4 structures combined with the discovery of DNA helicases that unwind them has generated renewed interest in these non-canonical DNA secondary structures. Mutation of two FANC-J family helicases results in instability of endogenous G-rich sequences (Cheung et al., 2002
; London et al., 2008
). However, helicases function in RNA transcription, processing and translation, not just DNA mechanics, and the FANC-J studies do not allow the conclusion that these helicases act directly on G-rich or G4 structures. To our knowledge, this is the first demonstration of a DNA helicase having a direct role at G4 motifs in vivo
. We also provide evidence for a mechanistic basis for instability of G4 motifs in the absence of a G4-resolving helicase.
G4 motifs were among the high confidence Pif1 binding sites (; q < 0.001), providing evidence that Pif1 acts directly on G4 motifs. However, consistent with its being a multi-functional helicase, G4 motifs were only a subset of Pif1 binding sites. In addition, only 25% of the G4 motifs were Pif1 associated. This measurement is an underestimate both because we used stringent criteria to identify binding sites and because we excluded the ~140 G4 motifs in telomeric DNA and the ~900 G4 motifs in rDNA, even though Pif1 bound extremely well to both (). It is also possible that not all G4 motifs form G4 structures or that their formation is limited to a time (e.g. meiosis) that was not monitored in our experiments or that different helicases act on different sets of G4 motifs. The demonstration that those G4 motifs that were Pif1 associated were more likely than other G4 motifs or other non G4 Pif1 binding sites to impede replication () and to stimulate DNA breakage () in pif1 cells makes a strong argument that their binding to Pif1 is biologically, as well as statistically, significant.
Unexpectedly, peak Pif1 binding to G4 motifs occurred after replication of the regions containing the motifs, although there was also significant binding to the motifs at earlier times in the cell cycle (). This binding pattern is consistent with the cell cycle regulated abundance of nuclear Pif1, which is present throughout the cell cycle but is maximally expressed in late S/G2 phase (Vega et al., 2007
). Perhaps Pif1 unwinds G4 motifs throughout the cell cycle but binds and unwinds G4 motifs again after replication as a sort of failsafe mechanism that makes sure that the genome is free of G4 structures prior to chromosome condensation. Alternatively, other yeast helicases that act mainly earlier in the cell cycle could contribute to resolution of G4 structures. Another possibility is that G4 structures do not need to be resolved for forks to move past them (see below), but they must be resolved prior to mitosis.
By two criteria, DNA Pol2 occupancy () and 2D gels (), replication forks slowed in the vicinity of G4 motifs in pif1-m2 cells. The genome-wide studies are particularly compelling as those G4 motifs that bound Pif1 were much more likely than non Pif1 binding G4 motifs to have high DNA Pol2 occupancy in pif1-m2 cells (q < 0.001 versus q = 0.47). Likewise, four of four tested G4 motifs that were Pif1 associated in WT cells stimulated recombination in the direct repeat assay in pif1Δ cells while three of three non G4 Pif1 binding sites did not (). Even more dramatically, when two G4 motifs were mutated so they no longer are predicted to form G4 structures but retained the same high GC content they had prior to mutation, neither of the mutated motifs bound Pif1 () or increased DNA Pol2 occupancy (), DNA breakage (), or recombination in the absence of Pif1 (). Thus, being Pif1 associated or being G-rich was not sufficient to affect replication or chromosome fragility in pif1 mutant cells.
The second unanticipated result from this study is that the replication fork slowing near G4 motifs in pif1-m2
cells was regional rather than site specific. This characteristic was evident from both 2D gels () and DNA Pol2 levels (). In contrast, stable protein complexes slow fork progression in a site-limited manner (Deshpande and Newlon, 1996
; Greenfeder and Newlon, 1992
; Ivessa et al., 2003
). It was harder to detect fork slowing by 2D gels than by ChIP, perhaps because the latter is more sensitive. Alternatively, and we think more likely, replication intermediates in the vicinity of a G4 motif were difficult to isolate intact, as required for 2D gels but not for ChIP, as their recovery required in vivo
psoralen cross-linking. This requirement suggests that while forks can ultimately bypass G4 structures, the DNA in the vicinity of the bypassed motifs is often damaged, containing nicks or gaps. Together these data indicate that forks slow in both their approach and movement away from a G4 motif, a behavior that suggests that unresolved G4 structures affect DNA topology/chromatin structure or generate torsional stress that acts over several kb.
In addition, Pif1 deficient, but not WT or sgs1Δ, cells were sensitive to large numbers of G4 motifs under conditions where replication was impaired by HU (). The slow growth of Pif1 deficient cells in HU was eliminated by spontaneous mutations in the G4 motifs (). The majority of these mutations were located in the G-residues of the G-tracts, and, in virtually all cases, these mutations eliminated the predicted ability of the insert to form a G4 structure. Three of three tested spontaneous mutations lost Pif1 binding and their negative effects on chromosome integrity concomitant with losing the ability to form a G4 structure. The frequent mutations in G residues within G4 motifs must confer a selective advantage that makes it easier to maintain high plasmid copy number.
It seems unlikely that all of Pif1’s effects are due to its unwinding G4 structures, especially as many of its binding sites were not G4 motifs. For example, 8% of its binding sites were at highly transcribed genes, most of which lack a G4 motif. Because Pif1 has the unusual property of being more active at displacing RNA than DNA from a DNA substrate (Boule and Zakian, 2007
), it might act at these genes by removing RNA from highly transcribed regions. However, other Pif1 functions might be related to unwinding G4 structures. For example, Pif1’s role in generating long flap Okazaki fragments might occur when a G4 structure forms in an extruded flap, and its resolution by Pif1 generates a longer than average length flap that requires Dna2 for processing (). Pif1’s critical but poorly understood role in mitochondrial DNA might also involve G4 structures, as remarkably, the very AT-rich yeast mitochondrial genome has a nearly tenfold higher density of G4 motifs than nuclear DNA (Capra et al., 2010
We end with a speculative model to explain our data (). We propose that growth in HU or reduced Pif1 increases the probability of G4 structure formation and/or persistence. If a G4 structure is present when the region containing it is replicating, forks slow as they approach (and move away from) the structure but are usually able to bypass the G4 structure, just as they bypass other DNA lesions, leaving nicks or gaps behind. More rarely, forks arrest at G4 structures in pif1-m2
cells (see converged forks; , arrows). If G4 structures are present at the end of S phase, Pif1 binds to and resolves them, and doing so suppresses breakage at these sites as chromosomes condense in preparation for mitosis. This model may be relevant to the function of human PIF, which also unwinds G4 structures in vitro
) since like yeast Pif1 (Vega et al., 2007
), human PIF is cell cycle regulated with peak abundance in late S/G2 phase (Mateyak and Zakian, 2006