Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

Abstract

RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. Here, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3′ single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ∼90°to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATP hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. This bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.helicase | RecQ | mechanism | aromatic-rich loop | DNA bending H elicases are motor proteins that convert the chemical energy of nucleoside triphosphate (NTP) hydrolysis into the mechanical energy needed to separate nucleic acid strands (1). The largest and most diverse helicase superfamilies, SF1 and SF2, use conserved sequence motifs (I, Ia, II-VI) within their helicase domains to couple NTP hydrolysis to conformational changes that mediate DNA translocation and unwinding (1, 2). Although the DNA-unwinding mechanisms of SF1 helicases have been examined extensively, far less is known about SF2 enzymes, in part because of the smaller number of available helicase/substrate complex structures.RecQ DNA helicases are SF2 enzymes with broad roles in promoting genomic stability in eubacterial and eukaryotic species (3). Their importance is underscored by the multiple genomic instability diseases caused by mutations in human recQ genes (4-8). At a structural level, most RecQ helicases share a similar domain architecture that includes a helicase domain, a RecQ C-terminal (RQC) element comprised of Zn 2+ -binding and winged-helix (WH) domains, and a helicase and RNaseD C-terminal (HDRC) domain (Fig. 1A) (9, 10). The helicase and RQC domains combine to form a catalytic core that is sufficient for DNA-unwinding activity in many RecQ proteins. Crystal structures of several RecQ catalytic cores have been determined, including Escherichia coli RecQ (EcRecQ), human RecQ1, and human Bloom syndrome protein (BLM) [refs. 10-12 and unpublished structures (4CDG, 2WWY, and 4CGZ) available through the Protein Data Bank (PDB)]. A comparison of these structures reveals strong similarities among domains within the catalytic core but also differences in the relative positioning of these domains among RecQ proteins. The EcRecQ and antibody-bound BLM structures form an open arrangement in which the WH domain is centered relative to the helicase and Zn 2+ -binding domains, but in RecQ1 and DNA-bound BLM a closed arrangement is observed with the WH domain positioned laterally to the helicase domain (Fig. 1B and Fig. S1) (10-12). The functional relevance of...

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding

“…Unlike helicases in replication, RecQ requires neither initiation factors nor a specific sequence to melt DNA. Similar to Orc1, however, RecQ has a winged-helix domain that is thought to recognize specific DNA structures (18,63). Because we observe a dimer of RecQ initiating unwinding, it is possible that, again similar to Orc1, the winged-helix domain of two monomers of RecQ may bind to and distort dsDNA to initiate unwinding.…”

“…Alternatively, because RecQ can bind dsDNA, albeit with lower affinity than ssDNA (7), two RecQ monomers might bind dsDNA, and induce an open melted conformation of the DNA. The energetic aspects of this model are supported by a recent crystal structure of Cronobacter sakazakii RecQ (CsRecQ), which is 86% identical to E. coli RecQ (18). In a cocomplex with DNA, CsRecQ is observed to separate 2 bp of DNA at the ssDNA/dsDNA junction in the absence of ATP, showing that the binding energy of one RecQ monomer is sufficient to melt dsDNA.…”

“…The wingedhelix domain of RecQ is important for the recognition of this broad array of DNA substrates (18). This domain binds to dsDNA yet it adopts a flexible conformation that allows it to adapt to many DNA structures.…”

Human RECQ1 helicase-driven DNA unwinding, annealing, and branch migration: Insights from DNA complex structures

“…The cleft between the domains is at its most open state, which may allow the release of the hydrolyzed nucleotide (and exchange for ATP). A second conformation, in which both the D1 and the D2 domains are tightly attached to the DNA at adjacent sites, may be represented by a structure of a DNA-bound RecQ protein from the bacterium C. sakazakii (PDB ID code 4TMU) (21). That structure maintains the highly conserved ratchet structure around the ssDNA close to the branch point: Specifically, residues R324, A346, T371, and K393 in RECQ1 are matched by the equivalent R246, A268, T293, and R315 in C. sakazakii RecQ (CsRecQ).…”

“…Crucially, the ssDNA chain binds tightly to the ARL immediately next to the D2 contact, followed by tight binding to helicase motifs Ia and Ib. Although this structure does not contain ATP, Manthei et al (21) argue that it represents a conformation poised for ATP hydrolysis. Thus, it seems that the two structures represent two extreme states of the catalytic cycle.…”