Rapid unwinding of triplet repeat hairpins by Srs2 helicase of Saccharomyces cerevisiae

Abstract

Expansions of trinucleotide repeats cause at least 15 heritable human diseases. Single-stranded triplet repeat DNA in vitro forms stable hairpins in a sequence-dependent manner that correlates with expansion risk in vivo. Hairpins are therefore considered likely intermediates during the expansion process. Unwinding of a hairpin by a DNA helicase would help protect against expansions. Yeast Srs2, but not the RecQ homolog Sgs1, blocks expansions in vivo in a manner largely dependent on its helicase function. The current study tested the idea that Srs2 would be faster at unwinding DNA substrates with an extrahelical triplet repeat hairpin embedded in a duplex context. These substrates should mimic the relevant intermediate structure thought to occur in vivo. Srs2 was faster than Sgs1 at unwinding several substrates containing triplet repeat hairpins or another structured loop. In contrast, control substrates with an unstructured loop or a Watson–Crick duplex were unwound equally well by both enzymes. Results with a fluorescently labeled, three-way junction showed that Srs2 unwinding proceeds unabated through extrahelical triplet repeats. In summary, Srs2 maintains its facile unwinding of triplet repeat hairpins embedded within duplex DNA, supporting the genetic evidence that Srs2 is a key helicase in Saccharomyces cerevisiae for preventing expansions.

Molecular Mechanism of Resolving Trinucleotide Repeat Hairpin by Helicases

“…We posit that the weak unwinding activity of Srs2 enables it to primarily focus on destabilizing the TNR hairpin rather than proceeding to dsDNA unwinding. This is in agreement with the previous studies reporting that Srs2 is more efficient at resolving TNR than Sgs1 (Anand et al, 2012; Bhattacharyya and Lahue, 2004; Dhar and Lahue, 2008). The repetitive TNR unfolding activity exhibited by Srs2 may provide an efficient mechanism for allowing replication fork to proceed in the presence of DNA secondary structures.…”

“…We prepared an open-ended TNR hairpin similar to a previous study (Dhar and Lahue, 2008) where the hairpin consists of 7 repeats of CTG, with a short, 9 base pair dsDNA to hold the end of the hairpin stem closed (Figure 3A). The 9 bp duplex was inserted at the end of TNR only to hold the two strands together, not requiring an active unwinding activity.…”

“…Sgs1 unfolds the TNR hairpin in the same manner that it unwinds the duplex DNA. By adopting the open-ended TNR hairpin structure (Dhar and Lahue, 2008), we showed that Sgs1 does not bypass the TNR hairpin, but unwinds it, likely by tracking the single strand DNA from 3’ to 5’ direction (Bennett et al, 1999; Cejka and Kowalczykowski, 2010; Sun et al, 1999). In contrast, we observe that Srs2 remains at the site of the hairpin while repeatedly unfolding the TNR structure.…”

“…The genetic screening performed in Saccharomyces Cerevisiae revealed that DNA helicases Srs2 and Sgs1 are potential inhibitors of TNR expansions (Anand et al, 2012; Bhattacharyya and Lahue, 2004; Dhar and Lahue, 2008; Kerrest et al, 2009). Consistently, cells lacking the gene for Srs2 showed a significant increase (up to 40 folds) in TNR expansions and contractions, resulting in chromosomal fragility (Anand et al, 2012; Bhattacharyya and Lahue, 2004; Kerrest et al, 2009).…”

Multifunctional Roles of Saccharomyces cerevisiae Srs2 protein in Replication, Recombination and Repair

“…4). Both in vivo and in vitro Srs2 can unwind triplet repeat hairpins (Bhattacharyya and Lahue 2004;Dhar and Lahue 2008;Anand et al 2012), permitting replication without expansion or contraction of repeats. In vitro, compared to a random 3 -overhanging duplex DNA substrate, purified Srs2 helicase displays higher efficiency unwinding or a similar 3 -overhanging DNA with a triplet-repeat hairpin embedded (Bhattacharyya and Lahue 2005).…”