RECQ1 is the shortest among the five human RecQ helicases comprising of two RecA like domains, a zinc-binding domain and a RecQ C-terminal domain containing the winged-helix (WH). Mutations or deletions on the tip of a β-hairpin located in the WH domain are known to abolish the unwinding activity. Interestingly, the same mutations on the β-hairpin of annealing incompetent RECQ1 mutant (RECQ1T1) have been reported to restore its annealing activity. In an attempt to unravel the strand annealing mechanism, we have crystallized a fragment of RECQ1 encompassing D2–Zn–WH domains harbouring mutations on the β-hairpin. From our crystal structure data and interface analysis, we have demonstrated that an α-helix located in zinc-binding domain potentially interacts with residues of WH domain, which plays a significant role in strand annealing activity. We have shown that deletion of the α-helix or mutation of specific residues on it restores strand annealing activity of annealing deficient constructs of RECQ1. Our results also demonstrate that mutations on the α-helix induce conformational changes and affects DNA stimulated ATP hydrolysis and unwinding activity of RECQ1. Our study, for the first time, provides insight into the conformational requirements of the WH domain for efficient strand annealing by human RECQ1.
The laser light scattering experiments were performed to explore the role of dextran (size (d): 2.6, 6.9, and 17.0 nm) in compacting the plasmids (pBS: 2.9 kbps; pCMV-Tag2B: 4.3 kbps; and pET28a: 5.3 kbps) in vitro in the volume fraction (ϕ) range 0.01 to 0.15 of the macromolecular crowder. Two compaction regimes were observed in terms of the radius of gyration (R g ) for plasmid−dextran combinations, wherein the plasmid diffusivity is governed by normal diffusion and subdiffusion, respectively. Generalized scaling, R g ∼ ϕ −1/(1+x) , where x represents the conformational geometry of plasmids, is reported. The plasmid conformation depends on the crowder's size, with larger conformational changes observed in the presence of smaller crowders. The second virial coefficient (A 2 ) and translational diffusion coefficient (D t ) indicate that entropically driven depletion of crowders, excluded volume, and interplasmid repulsive interactions govern plasmids' conformational changes, validated herein from the scaling of D t with molecular weight.
RecQ helicases are superfamily 2 (SF2) DNA helicases that unwind a wide spectrum of complex DNA structures in a 3 0 to 5 0 direction and are involved in maintaining genome stability. RecQ helicases from protozoan parasites have gained significant interest in recent times because of their involvement in cellular DNA repair pathways, making them important targets for drug development. In this study, we report biophysical and biochemical characterization of the catalytic core of a RecQ helicase from hemoflagellate protozoan parasite Leishmania donovani. Among the two putative RecQ helicases identified in L. donovani, we cloned, overexpressed and purified the catalytic core of LdRECQb. The catalytic core was found to be very efficient in unwinding a wide variety of DNA substrates like forked duplex, 3 0 tailed duplex and Holliday junction DNA. Interestingly, the helicase core also unwound blunt duplex with slightly less efficiency. The enzyme exhibited high level of DNA-stimulated ATPase activity with preferential stimulation by forked duplex, Holliday junction and 3 0 tailed duplex. Walker A motif lysine mutation severely affected the ATPase activity and significantly affected unwinding activity. Like many other RecQ helicases, L. donovani RECQb also possesses strand annealing activity. Unwinding of longer DNA substrates by LdRECQb catalytic core was found to be stimulated in the presence of replication protein A (LdRPA-1) from L. donovani. Detailed biochemical characterization and comparison of kinetic parameters indicate that L. donovani RECQb shares considerable functional similarity with human Bloom syndrome helicase.
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