Telomeres do the (un)twist: helicase actions at chromosome termini

Abstract

Telomeres play critical roles in protecting genome stability, and their dysfunction contributes to cancer and age-related degenerative diseases. The precise architecture of telomeres, including their single-stranded 3' overhangs, bound proteins, and ability to form unusual secondary structures such as t-loops, is central to their function and thus requires careful processing by diverse factors. Furthermore, telomeres provide unique challenges to the DNA replication and recombination machinery, and are particularly suited for extension by the telomerase reverse transcriptase. Helicases use the energy from NTP hydrolysis to track along DNA and disrupt base pairing. Here we review current findings concerning how helicases modulate several aspects of telomere form and function.

Figure 1

Potential secondary structures at telomeres. A) Through the assistance of TRF2 and perhaps other factors, the 3′ overhang loops back and invades into internal telomere repeats forming a t-loop. This is thought to help protect the telomere terminus from further exonucleolytic processing and to prevent it from inappropriately activating checkpoint proteins. B) The general structures of a G-quartet (left) and of an intramolecular G-quadruplex (right) are shown. C) Illustration of a G-quadruplex that has formed at the 3′ overhang, although it is possible that G-quadruplexes also form among internal telomere repeats, particularly under conditions where they become single-stranded, e.g. replication and recombination.

Figure 2

Examples of helicase-assisted mechanisms of replication fork rescue. A) A replication fork stalls or collapses at an inhibitory lesions (black dot). The replication fork can then regress via reverse branch migration to form a “chicken foot” structure, followed by copying of the newly synthesized sister strand to generate sequence beyond the lesion. Dissolution of the regressed chicken foot occurs by reverse branch migration, and replication resumes. Helicases could be involved at the branch migration steps. B) A lesion that blocks replication is bypassed by a switch in template from the parental strand to the newly replicated sister chromatid. Once the lesion has been bypassed, reinvasion back to the parental template can occur, and then resolution of entwined strands allows the sister chromatids to separate. Helicases could assist in the original template switch, the reinvasion step, and the final resolution step. C) If a collapsed fork leads to a double strand break, resection of the broken DNA end to generate a recombinogenic 3′ end allows invasion of the intact chromatid and resumption of DNA synthesis. Branch migration of the D-loop (to the left) establishes a full Holliday junction, thus allowing the resumption of replication. Helicases could assist in end-processing, invasion and branch migration.

Figure 2

Examples of helicase-assisted mechanisms of replication fork rescue. A) A replication fork stalls or collapses at an inhibitory lesions (black dot). The replication fork can then regress via reverse branch migration to form a “chicken foot” structure, followed by copying of the newly synthesized sister strand to generate sequence beyond the lesion. Dissolution of the regressed chicken foot occurs by reverse branch migration, and replication resumes. Helicases could be involved at the branch migration steps. B) A lesion that blocks replication is bypassed by a switch in template from the parental strand to the newly replicated sister chromatid. Once the lesion has been bypassed, reinvasion back to the parental template can occur, and then resolution of entwined strands allows the sister chromatids to separate. Helicases could assist in the original template switch, the reinvasion step, and the final resolution step. C) If a collapsed fork leads to a double strand break, resection of the broken DNA end to generate a recombinogenic 3′ end allows invasion of the intact chromatid and resumption of DNA synthesis. Branch migration of the D-loop (to the left) establishes a full Holliday junction, thus allowing the resumption of replication. Helicases could assist in end-processing, invasion and branch migration.

Figure 3

End-processing of telomeres after replication. The G-rich strand is replicated by lagging strand synthesis, and even with fully efficient lagging strand synthesis the removal of the terminal RNA primer allows for the generation of a 3′ overhang. Helicases could assist with RNA primer removal and might also aid with additional nucleolytic processing. The C-rich strand is replicated via leading strand synthesis and therefore for the 3′ overhang to be generated, the activity of exonucleases and/or endonucleases such as the Mre11 are required, which could be assisted by helicases.

Figure 4

Depictions of the non-homologous end joining (NHEJ) and single-strand annealing (SSA) pathways of DSB repair. A) In NHEJ, double strand breaks are essentially re-ligated back together. B) In SSA, nucleolytic processing of DNA ends (perhaps dependent upon RecQ-family helicases) occurs, and regions of homology are utilized to help guide the ligation of DNA breaks. The non-homologous 3′ flaps are removed by nucleases, such as Rad1/10, which can be assisted by the Srs2 helicase when the flaps are long.

Figure 5

t-loop dynamics. A standard t-loop might be unwound by a helicase (e.g. WRN or BLM) to facilitate replication of the telomere. In addition, a helicase (e.g. WRN), perhaps working together with HR factors, could allow a t-loop to branch migrate to form a double HJ at its base. If resolved with crossing over (e.g. by a classical HJ resolvase or by the concerted action of nucleases), this could lead to telomere truncation and t-circle formation. BLM, together with Topo IIIα, can resolve a double HJ in the middle of extensive flanking sequences and could thus dissolve a double HJ t-loop. Also, because a double HJ t-loop is near an end, other helicases, such as WRN may be able to remove it by simple branch migration