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The RecQ family of helicases are important for maintenance of genomic integrity. Although functions of constructive subdomains of this family of helicases have been extensively studied, the helical hairpin (HH) in the RecQ-C-terminal domain (RQC) has been underappreciated and remains poorly understood. Here by using single-molecule fluorescence resonance energy transfer, we found that HH in the human BLM transiently intercepts different numbers of nucleotides when it is unwinding a double-stranded DNA. Single-site mutations in HH that disrupt hydrogen bonds and/or salt bridges between DNA and HH change the DNA binding conformations and the unwinding features significantly. Our results, together with recent clinical tests that correlate single-site mutations in HH of human BLM with the phenotype of cancer-predisposing syndrome or Bloom's syndrome, implicate pivotal roles of HH in BLM's DNA unwinding activity. Similar mechanisms might also apply to other RecQ family helicases, calling for more attention to the RQC helical hairpin.

G‐quadruplex (G4) is a four‐stranded noncanonical DNA structure that has long been recognized as a potential hindrance to DNA replication. However, how replisomes effectively deal with G4s to avoid replication failure is still obscure. Here, using single‐molecule and ensemble approaches, the consequence of the collision between bacteriophage T7 replisome and an intramolecular G4 located on either the leading or lagging strand is examined. It is found that the adjacent fork junctions induced by G4 formation incur the binding of T7 DNA polymerase (DNAP). In addition to G4, these inactive DNAPs present insuperable obstacles, impeding the progression of DNA synthesis. Nevertheless, T7 helicase can dismantle them and resolve lagging‐strand G4s, paving the way for the advancement of the replication fork. Moreover, with the assistance of the single‐stranded DNA binding protein (SSB) gp2.5, T7 helicase is also capable of maintaining a leading‐strand G4 structure in an unfolded state, allowing for a fraction of T7 DNAPs to synthesize through without collapse. These findings broaden the functional repertoire of a replicative helicase and underscore the inherent G4 tolerance of a replisome. [ABSTRACT FROM AUTHOR]

G-quadruplexes (G4s) are important in regulating DNA replication, repair and RNA transcription through interactions with specialized proteins. Dbp2 has been identified as a G4 DNA binding protein from Saccharomyces cerevisiae cell lysates. The majority of G4 motifs in Saccharomyces cerevisiae display 5–50 nt loops, only a few have 1–2 nt loops. Human DDX5 could unfold MycG4 DNA, whether Dbp2 also participates in remodeling G4 motifs with short loops in Saccharomyces cerevisiae remains elusive. Here we find that Dbp2 prefers G-rich substrates and binds MycG4 with a high affinity. Dbp2 possesses a dual function for different conformations of MycG4, destabilizing the folded MycG4 and inducing further folding of the unfolded MycG4. Similarly, DDX5 can unfold MycG4, but it exhibits a weaker MycG4 folding-promoting activity relative to Dbp2. Furthermore, Dbp2 facilitates DNA annealing activity in the absence of ATP, suggesting that Dbp2 can work on DNA substrates and possibly participate in DNA metabolism. Our results demonstrate that Dbp2 plays an important role in regulating the folding and unfolding activities of MycG4. • Dbp2 prefers G-rich ssDNA and ssRNA substrates. • Dbp2 destabilized the folded MycG4 DNA. • Dbp2 induced further folding of the unfolded MycG4 DNA. • RNA helicase Dbp2 could anneal DNA and DNA substrates. [ABSTRACT FROM AUTHOR]

The RecQ family of helicases are important for maintenance of genomic integrity. Although functions of constructive subdomains of this family of helicases have been extensively studied, the helical hairpin (HH) in the RecQ-C-terminal domain (RQC) has been underappreciated and remains poorly understood. Here by using single-molecule fluorescence resonance energy transfer, we found that HH in the human BLM transiently intercepts different numbers of nucleotides when it is unwinding a double-stranded DNA. Single-site mutations in HH that disrupt hydrogen bonds and/or salt bridges between DNA and HH change the DNA binding conformations and the unwinding features significantly. Our results, together with recent clinical tests that correlate single-site mutations in HH of human BLM with the phenotype of cancer-predisposing syndrome or Bloom's syndrome, implicate pivotal roles of HH in BLM's DNA unwinding activity. Similar mechanisms might also apply to other RecQ family helicases, calling for more attention to the RQC helical hairpin., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

I-motifs are noncanonical four-stranded DNA structures formed by C-rich sequences at acidic environment with critical biofunctions. The particular pH sensitivity has inspired the development of i-motifs as pH sensors and DNA motors in nanotechnology. However, the folding and regulation mechanisms of i-motifs remain elusive. Here, using single-molecule FRET, we first show that i-motifs are more dynamic than G4s. Impressively, i-motifs display a high diversity of six folding species with slow interconversion. Further results indicate that i-motifs can be linearized by Replication protein A. More importantly, we identified a number of helicases with high specificity to i-motifs at low pH. All these helicases directly act on and efficiently resolve i-motifs into intermediates independent of ATP, although they poorly unwind G4 or duplex at low pH. Owing to the extreme sensitivity to helicases and no need for ATP, i-motif may be applied as a probe for helicase sensing both in vitro and in vivo ., Competing Interests: The authors declare no competing financial interest., (© 2021 The Author(s).)