Abstract
Various helicases and single-stranded DNA (ssDNA) binding proteins are known to destabilize G-quadruplex (GQ) structures, which otherwise result in genomic instability. Bulk biochemical studies have shown that Bloom helicase (BLM) unfolds both intermolecular and intramolecular GQ in the presence of ATP. Using single molecule FRET, we show that binding of RecQ-core of BLM (will be referred to as BLM) to ssDNA in the vicinity of an intramolecular GQ leads to destabilization and unfolding of the GQ in the absence of ATP. We show that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to ssDNA overhang, as modulated by the nucleotide state, ionic conditions, overhang length and overhang directionality. In particular, we observed enhanced GQ unfolding by BLM in the presence of non-hydrolysable ATP analogs, which has implications for the underlying mechanism. We also show that increasing GQ stability, via shorter loops or higher ionic strength, reduces BLM-mediated GQ unfolding. Finally, we show that while WRN has similar activity as BLM, RecQ and RECQ5 helicases do not unfold GQ in the absence of ATP at physiological ionic strength. In summary, our study points to a novel and potentially very common mechanism of GQ destabilization mediated by proteins binding to the vicinity of these structures.
Highlights
Human Bloom helicase (BLM) is a member of RecQ family [1,2], which includes Escherichia coli RecQ, Saccharomyces cerevisiae Sgs1p, Schizosaccharomyces pombe Rqh1 and human WRN, RECQL1, RECQL4 and RECQL5 helicases [3,4,5,6,7]
These measurements yielded consistent results with those on pd-hGQ12T and showed that BLM-mediated G-quadruplex structures (GQs) unfolding is most efficient in the adenosine triphosphate (ATP)␥ S state, followed by the nt-free state and the ADP state (Supplementary Figure S14)
The data presented far show that BLM-mediated GQ unfolding is most efficient in the ATP␥ S state, followed by the nt-free and ADP states
Summary
Human Bloom helicase (BLM) is a member of RecQ family [1,2], which includes Escherichia coli RecQ, Saccharomyces cerevisiae Sgs1p, Schizosaccharomyces pombe Rqh and human WRN, RECQL1, RECQL4 and RECQL5 helicases [3,4,5,6,7]. BLM has been shown to form a hexameric ring [10] and other multimeric structures [11]. BLM mutants lacking the oligomerization domain can unwind dsDNA, in the 3′ to 5′ direction [12]. Even though BLM is capable of forming multimeric structures, recent studies suggest that such structures dissociate upon ATP hydrolysis and it is the BLM monomer that unwinds dsDNA and resolves non-canonical DNA structures, such as Holliday junctions and D-loops [11]
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