RecQ DNA helicases are critical for proper maintenance of genomic stability and mutations in multiple human RecQ genes are linked with genetic disorders characterized by a predisposition to cancer. RecQ proteins are conserved from prokaryotes to humans and in all cases form higher-order complexes with other proteins to efficiently execute their cellular functions. The focus of this review is a conserved complex that is formed between RecQ helicases and type-I topoisomerases. In humans, this complex is referred to as the BLM dissolvasome or BTR complex, and is comprised of the RecQ helicase BLM, topoisomerase IIIα, and the RMI proteins. The BLM dissolvasome functions to resolve linked DNA intermediates without exchange of genetic material, which is critical in somatic cells. We will review the history of this complex and highlight its roles in DNA replication, recombination, and repair. Additionally, we will review recently established interactions between BLM dissolvasome and a second set of genome maintenance factors (the Fanconi anemia proteins) that appear to allow coordinated genome maintenance efforts between the two systems.

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...

Lecithin:cholesterol acyltransferase (LCAT) plays a key role in reverse cholesterol transport by transferring an acyl group from phosphatidylcholine to cholesterol, promoting the maturation of high-density lipoproteins (HDL) from discoidal to spherical particles. LCAT is activated through an unknown mechanism by apolipoprotein A-I (apoA-I) and other mimetic peptides that form a belt around HDL. Here, we report the crystal structure of LCAT with an extended lid that blocks access to the active site, consistent with an inactive conformation. Residues Thr-123 and Phe-382 in the catalytic domain form a latch-like interaction with hydrophobic residues in the lid. Because these residues are mutated in genetic disease, lid displacement was hypothesized to be an important feature of apoA-I activation. Functional studies of site-directed mutants revealed that loss of latch interactions or the entire lid enhanced activity against soluble ester substrates, and hydrogen-deuterium exchange (HDX) mass spectrometry revealed that the LCAT lid is extremely dynamic in solution. Upon addition of a covalent inhibitor that mimics one of the reaction intermediates, there is an overall decrease in HDX in the lid and adjacent regions of the protein, consistent with ordering. These data suggest a model wherein the active site of LCAT is shielded from soluble substrates by a dynamic lid until it interacts with HDL to allow transesterification to proceed.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes a critical step of reverse cholesterol transport by esterifying cholesterol in high density lipoprotein (HDL) particles. LCAT is activated by apolipoprotein A-I (ApoA-I), which forms a double belt around HDL, however the manner in which LCAT engages its lipidic substrates and ApoA-I in HDL is poorly understood. Here, we used negative stain electron microscopy, crosslinking, and hydrogendeuterium exchange studies to refine the molecular details of the LCAT-HDL complex. Our data are consistent with LCAT preferentially binding to the edge of discoidal HDL near the boundary between helix 5 and 6 of ApoA-I in a manner that creates a path from the lipid bilayer to the active site of LCAT. Our results provide not only an explanation why LCAT activity diminishes as HDL particles mature, but also direct support for the anti-parallel double belt model of HDL, with LCAT binding preferentially to the helix 4/6 region.

Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structure, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a cDNA hairpin. It is not clear how NC specifically destabilizes TAR RNA but does not strongly destabilize the resulting annealed RNA-DNA hybrid structure, which must be formed for reverse transcription to continue. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium TAR stability and unfolding barrier for TAR RNA. Experiments show that adding NC lowers the transition state barrier height while also dramatically shifting the barrier location. Incorporating TAR destabilization by NC into the mfold-based model reveals that a subset of preferential protein binding sites is responsible for the observed changes in the unfolding landscape, including the unusual shift in the transition state. We measure the destabilization induced at these NC binding sites and find that NC preferentially targets TAR RNA by binding to specific sequence contexts that are not present on the final annealed RNA-DNA hybrid structure. Thus, specific binding alters the entire RNA unfolding landscape, resulting in the dramatic destabilization of this specific structure that is required for reverse transcription.single molecule | force spectroscopy | RNA stretching | RNA binding T he transactivation response (TAR) RNA hairpin is a 59-nt sequence in the long-terminal repeat (LTR) of the HIV-1 genome that forms a 24-bp hairpin ( Fig. 1A) (1). This structure is essential in promoting viral transactivator protein (Tat)-mediated transcription. The protein-RNA complex further enhances LTR promoter activity (2). The highly stable TAR hairpin structure that stimulates viral RNA transcription becomes a liability during the early stage of a new infection, as TAR hairpins inhibit the minus-strand transfer step required for reverse transcription (1). To alleviate this inhibition, successful reverse transcription requires a key viral chaperone, the nucleocapsid (NC) protein. In vitro experiments have shown a 3,000-fold stimulation of the rate-limiting step of minus-strand transfer in the presence of NC (3), as NC is required to destabilize TAR RNA and the complementary repeat TAR DNA hairpin to allow subsequent strand annealing (1).HIV-1 NC is only 55 aa long, consisting of two highly conserved CCHC zinc fingers and a basic N terminus (1) (Fig. 1B). The multiple roles of NC during reverse transcription all use the same "chaperone" activity (1), which describes HIV-1 NC's ability to facilitate the rearrangement of nucleic acids into the most stable structures, with the lowest free energy (1). This chaperone activity is characterized by nucleic acid aggregation, duplex destabilization, and rapid kinetics of protein-nucleic acid interactions (3, 4). Aromatic residues in...