Single-molecule imaging reveals a common mechanism shared by G-quadruplex–resolving helicases

Abstract

G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power.

Structure and function of Pif1 helicase

“…Because the helicase remains anchored at the 3′-ssDNA junction, a loop of ssDNA is created as the helicase reels in the tail as it translocates toward the 3′-end of the DNA. Similar repetitive activity has been observed previously for E. coli Rep [91], E. coli UvrD [92], B. stearothermophilus PcrA [93], S. cerevisiae Srs2 [94], and human FANCJ [95], RHAU [96], WRN [96], and BLM [96,97] helicases. Such an activity could have biological significance in clearing DNA of proteins and secondary structures.…”

“…This mechanism has been suggested to be conserved among many helicases [96]. However, it is not known whether patrolling happens in vivo or how patrolling occurs.…”

The Chd1 Chromatin Remodeler Shifts Nucleosomal DNA Bidirectionally as a Monomer

“…For both 601 and 603 nucleosomes, Chd1 appeared unable to stably shift nucleosomes back to the starting position, exhibiting a highly repetitive sliding behavior (Figure 3 and S1). While this repetitive sliding may have been exacerbated by the absence of a C-terminal Chd1 domain of unknown function (Mohanty et al, 2016) that was absent in our construct, we note that many helicases translocate on nucleic acids in a highly repetitive manner (Koh et al, 2014; Myong et al, 2007; Myong et al, 2009; Myong and Ha, 2010; Myong et al, 2005; Park et al, 2010; Qiu et al, 2013; Tippana et al, 2016). Analogous to keeping nucleic acids unwound, repetitive sliding by Chd1 may keep nucleosomes in alternative states.…”

A direct view of the complex multi-pathway folding of telomeric G-quadruplexes

“…Although the majority of observed folded states are only marginally stable in current conditions, we expect that they can be individually recognized and trapped through selective structure-specific interactions with different ligands (62) and proteins (63). The structural knowledge available through our experiments can potentially be used to control the folding pathway of G4 structures, i.e.…”