Role and Regulation of Pif1 Family Helicases at the Replication Fork

Abstract

Pif1 helicases are a multifunctional family of DNA helicases that are important for many aspects of genomic stability in the nucleus and mitochondria. Pif1 helicases are conserved from bacteria to humans. Pif1 helicases play multiple roles at the replication fork, including promoting replication through many barriers such as G-quadruplex DNA, the rDNA replication fork barrier, tRNA genes, and R-loops. Pif1 helicases also regulate telomerase and promote replication termination, Okazaki fragment maturation, and break-induced replication. This review highlights many of the roles and regulations of Pif1 at the replication fork that promote cellular health and viability.

Keywords: DNA helicase; G-quadruplexes; Pif1 helicase; replication fork; replication fork barriers; telomerase.

Figure 1

Structure of BaPif1 (PDB ID: 5FHE [17]). The Pif1 signature motif (blue) provides structural support for the strand separation wedge (pink). The signature motif and wedge are insertions within domain 1A (green). Domains 1A and 2A (gray) are RecA-like domains that are conserved in all helicases. The SH3 domain (cyan) is an insertion within domain 2A. The bound DNA is yellow and ADP-AlF₄⁻ is orange.

Figure 2

Structure of BaPif1 bound to a forked DNA. (A) Two molecules of BaPif1 (tan and green) are bound to the DNA fork (magenta). (B) The BaPif1 on the 3′-arm of the fork (green) is bound at the junction. (C) The two BaPif1 molecules interact. PDB ID 6L3G. Images reproduced from reference [26] Creative Commons CC BY.

Figure 3

ScPif1 stimulates replication through lagging-strand G-quadruplexes. The leading strand is synthesized by Polε (yellow) in complex with PCNA (purple) at the same time as duplex unwinding by CMG (green). RPA (brown) may not be able to prevent G4 structures from forming on the lagging strand, so Pif1 (orange) may unfold G4 structures so that Polδ (pink) can synthesize the lagging strand in complex with PCNA. Since Polδ translocates 3′-to-5′ on the template and Pif1 translocates 5′-to-3′, Pif1 would approach the G4 structure from the opposite side to that of Polδ.

Figure 4

The role of Pif1 at R-loops may be to resolve G4 structures that form on the displaced strand. (A) Multiple G4 structures can form on the displaced strand of a telomeric R-loop. (B) G4 formation on the displaced strand of a co-transcriptional R-loop is likely to be more limited. In either case, Pif1 (orange triangle) is proposed to resolve the G4 structures on the displaced strand instead of resolving the R-loop itself. Green color shows RNA polymerase.

Figure 5

Extension of the 5′-flap of an Okazaki fragment by Pif1 provides a binding site for RPA that makes it resistant to cleavage by FEN1.

Figure 6

Pif1 promotes break induces replication with Polδ and PCNA. A second Pif1 may unwind the nascent strand to resolve the D-loop and reduce topological stress.

Figure 7

Pif1 promotes synthesis through G4 sequences by yeast and human mitochondrial polymerases. The role of Pif1 may be to unfold G4 structures that fold after the unwinding of the duplex before synthesis occurs.