Getting it done at the ends: Pif1 family DNA helicases and telomeres

Abstract

It is widely appreciated that the ends of linear DNA molecules cannot be fully replicated by the conventional replication apparatus. Less well known is that semi-conservative replication of telomeric DNA also presents problems for DNA replication. These problems likely arise from the atypical chromatin structure of telomeres, the GC-richness of telomeric DNA that makes it prone to forming DNA secondary structures, and from RNA-DNA hybrids, formed by transcripts of one or both DNA strands. Given the different aspects of telomeres that complicate their replication, it is not surprising that multiple DNA helicases promote replication of telomeric DNA. This review focuses on one such class of DNA helicases, the Pif1 family of 5'-3' DNA helicases. In budding and fission yeasts, Pif1 family helicases impact both telomerase-mediated and semi-conservative replication of telomeric DNA as well as recombination-mediated telomere lengthening.

Keywords: ALT; Break induced replication; DNA replication; Helicase; Pif1; TERRA; Telomerase; Telomere.

Fig 1

Telomere structures [84]. (A) Structure of telomeric DNA and telomere binding proteins in yeasts and humans. Not all proteins are shown and drawings are not to scale. Budding yeast telomeres consists of ~300 bp of an irregular sequence, 5′ -TG(1–3)-3′ ending in a ~12 nt long G-tail during most of the cell cycle. Rap1 binds duplex telomeric DNA and Cdc13 binds the G-tail. Rif1, Rif2, Sir2, Sir3, Sir4, Stn1, and Ten1 bind via protein–protein interactions. Fission yeast telomeres consist of ~250 bp of an irregular sequence, 5′ -G_(0–6)G₂T₂ACAC-3′ (the terminal C is present in ~13% of repeats). Taz1 binds duplex telomeric DNA and Pot1 binds the G-tail. Rap1, Rif1, Tpz1, Ccq1, and Poz1 bind via protein–protein interactions. At birth, human telomeres have ~15 kb of 5′ -TTAGGG-3′ repeats. TRF1 and TRF2 bind duplex telomeric DNA while the 3′ single-stranded G-tail (~100 nt long) is bound by POT1. Alternatively, the G-tail can be folded into a T-loop (as in panel C). RAP1, TIN1, and TPP1 bind via protein–protein interactions. (B) G-quadruplex structure [25]. The 3′ G-tail can form a stable four-stranded DNA structure, called a G-quadruplex (G4), held together by G–G Hoogsteen base-pairing. An intra-molecular G4 structure is shown. G4 structures can also form between the G-tails on different telomeres. (C) T-loop structure [85]. The 3′ overhang forms a lariat-like structure by the G-tail invading the adjacent duplex telomeric DNA. An internal G-strand bubble forms from the displaced strand that can be bound by POT1.

Fig. 2

Mechanisms for replicating chromosome ends. (A) Replicative DNA polymerases cannot replicate the very ends of linear DNA molecules. Owing to the properties of conventional DNA polymerases, removal of the terminal RNA primer leaves a gap at the 5′ ends of newly replicated strands. Black lines: parental strands of the DNA molecule; Blue rectangles: 8–12 nucleotide RNA primers; Green lines: Okazaki fragments; Orange lines: leading strand synthesis. Only the right end of a chromosome is shown. (B) In most eukaryotes, telomerase compensates for the loss of terminal sequences. Telomerase extends the 3′ end of a vertebrate telomere. Orange oval: TERT, the reverse transcriptase subunit of telomerase: Purple: Telomerase RNA and its template region; Green line: newly made telomeric DNA. (C) Telomeric DNA can be maintained by recombination or ALT (alternative lengthening of telomeres). Shown is break-induced-replication (BIR), which can repair a one-ended DNA break such as an eroded telomere. Red: newly synthesized DNA. The exact mechanism of BIR is still under discussion. Shown is a model where BIR is carried out by a migrating D-loop or bubble.

Fig. 3

Semi-conservative replication through telomeric DNA. (A) Replication fork moving towards the right telomere of a chromosome. Fork progression slowed by protein complexes (top), G-quadruplex DNA (middle), TERRA RNA (bottom). (B) Replication forks slow as they move through an internal tract of telomeric DNA in budding yeast in vivo (adapted from Ref. [8]). Three ~270 bp tracts of TG(1–3) DNA cloned from a telomere and separated from each other by a polylinker were inserted far from a telomere. Top: diagram of the construct; Bottom: two-dimension gel analysis of replication through the tracts. Accumulation of replication intermediates at telomeric tracts (indicated by bracket) is reflected by higher intensity of hybridization signal.