Break-Induced Replication Requires DNA Damage-Induced Phosphorylation of Pif1 and Leads to Telomere Lengthening

Abstract

Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR). Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway – Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening.

A Monomer of Pif1 Unwinds Double-Stranded DNA and It Is Regulated by the Nature of the Non-Translocating Strand at the 3′-End

“…Pif1 238–780 has a higher dissociation rate). Also, we generated Pif1 238–859 with phosphomimetic changes at two positions within the TLSS motif at the C-terminus (Figure S1), shown to be important for the activity of Pif1 in DSB repair and BIR 22,27 . Pif1 238–859/T763D–S766D unwinds with a similar rate (49 bp/sec) but to a lower extent than Pif1 238–859 , suggesting that introducing two negative charges in this region is sufficient to affect the processivity of Pif1.…”

“…The activity of Pif1 on de novo telomere addition is via a direct effect on the telomerase 23 and it requires phosphorylation of Thr763 and Ser766 in the TLSS motif at the C-terminus of the protein 27 . Phosphorylation of this motif has also been shown to be important for the activity of Pif1 in BIR 22 .…”

“…In contrast to this, it has also been shown that in the absence of Pif1 replication fork progression across either G4- or hairpin-forming DNA sequences is unaffected, rather it has been suggested that Pif1 might promotes replication fork movement across a DNA protein barrier 19 . Recently, it has been shown that Pif1 has a role in promoting DNA synthesis during break-induced replication 20 and in crossover recombination 20–22 . In vitro Pif1 stimulates extension by DNA polymerase δ of a Rad51-generated D-loop 20 .…”

Role of the Pif1-PCNA Complex in Pol δ-Dependent Strand Displacement DNA Synthesis and Break-Induced Replication

“…Pif1 possesses a conserved helicase core, and its N-terminal region (amino acids 40–233) (Figure 1A) appears to be unstructured and is dispensable for the helicase function (Singh et al, 2016). The C-terminal region of Pif1 (amino acids 788–859) (Figure 1A) is a target of Mec1-Rad53-induced phosphorylation for the regulation of telomerase at DSBs (Makovets and Blackburn, 2009; Vasianovich et al, 2014). Pif1 physically interacts with PCNA (Wilson et al, 2013), but the Pif1 region that mediates the interaction is not known.…”

Replication fork instability and the consequences of fork collisions from rereplication

“…These results support the hypothesis that BIR establishes processive replication forks in the absence of an origin. In addition to canonical fork requirements, the appearance of BIR repair products depends on the nonessential Pol δ subunit Pol32 (Lydeard et al 2007), and the Pif1 helicase is required for long-range synthesis during BIR (Saini et al 2013;Wilson et al 2013;Vasianovich et al 2014).…”