DNA Replication through G-Quadruplex Motifs Is Promoted by the Saccharomyces cerevisiae Pif1 DNA Helicase

Abstract

G-quadruplex (G4) DNA structures are extremely stable four-stranded secondary structures held together by non-canonical G-G base pairs. Genome-wide chromatin immuno-precipitation was used to determine the in vivo binding sites of the multi-functional S. cerevisiae Pif1 DNA helicase, a potent unwinder of G4 structures in vitro. G4 motifs were a significant subset of the high confidence Pif1 binding sites. Replication slowed in the vicinity of these motifs, and they were prone to breakage in Pif1-deficient cells, while non G4 Pif1 binding sites did not show this behavior. Introducing many copies of G4 motifs caused slow growth in replication stressed Pif1 deficient cells that was relieved by spontaneous mutations that eliminated their ability to form G4 structures, bind Pif1, slow DNA replication and stimulate DNA breakage. These data suggest that G4 structures form in vivo and that they are resolved by Pif1 to prevent replication fork stalling and DNA breakage.

Replication of Telomeres and the Regulation of Telomerase

“…Indeed, in mice, the Wrn phenotype, which is characterized by multiple signs of premature aging, only manifests itself upon concomitant deletion of telomerase (Chang et al 2004). S. cerevisiae Pif1 is another DNA helicase important for replication of Grich sequences being enriched at telomeres and other G-rich sequences throughout the genome that may form G-quadruplex structures (Paeschke et al 2011).…”

A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites

“…This human pathogen evades its host's immune system by changing the identity of its surface antigen via a G4 DNAinduced recombination event 10 . Importantly, however, this example also illustrates the recombinogenic and thus potentially pathological nature of this highly thermal stable non-B-DNA structure-a concern further fuelled by the notion of hampered DNA replication near G4 sequences in yeast [11][12][13] and the observed association of G4 motifs with structural genomic variations in human cancers 14 .…”

A quantitative telomeric chromatin isolation protocol identifies different telomeric states

“…These proteins also perform crucial functions, a fact that is underlined by the finding that they are mutated in several fatal degenerative syndromes. Some of them such as the Werner protein, Rtel1 and Pif1 are involved in the semiconservative DNA replication of telomeres for example to unwind G-quadruplex DNA structures, in which guanine bases of the telomeric G-strand associate through Hoogsteen hydrogen bonding to form tetrads [11][12][13][14] . Other important telomere factors are involved in the regulation of telomerase.…”