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Cutting antiparallel DNA strands in a single active site.

ABSTRACT: A single enzyme active site that catalyzes multiple reactions is a well-established biochemical theme, but how one nuclease site cleaves both DNA strands of a double helix has not been well understood. In analyzing site-specific DNA cleavage by the mammalian RAG1-RAG2 recombinase, which initiates V(D)J recombination, we find that the active site is reconfigured for the two consecutive reactions and the DNA double helix adopts drastically different structures. For initial nicking of the DNA, a locally unwound and unpaired DNA duplex forms a zipper via alternating interstrand base stacking, rather than melting as generally thought. The second strand cleavage and formation of a hairpin-DNA product requires a global scissor-like movement of protein and DNA, delivering the scissile phosphate into the rearranged active site.

SUBMITTER: Chen X

PROVIDER: S-EPMC7015813 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Cutting antiparallel DNA strands in a single active site.

Chen Xuemin X Cui Yanxiang Y Best Robert B RB Wang Huaibin H Zhou Z Hong ZH Yang Wei W Gellert Martin M

Nature structural & molecular biology 20200203 2

A single enzyme active site that catalyzes multiple reactions is a well-established biochemical theme, but how one nuclease site cleaves both DNA strands of a double helix has not been well understood. In analyzing site-specific DNA cleavage by the mammalian RAG1-RAG2 recombinase, which initiates V(D)J recombination, we find that the active site is reconfigured for the two consecutive reactions and the DNA double helix adopts drastically different structures. For initial nicking of the DNA, a lo ...[more]

PMID: 32015552

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Project description:Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. However, knowledge of the intact human telomeric G-quadruplex structure(s) formed under physiological conditions is a prerequisite for structure-based rational drug design. Here we report the folding structure of the human telomeric sequence in K+ solution determined by NMR. Our results demonstrate a novel, unprecedented intramolecular G-quadruplex folding topology with hybrid-type mixed parallel/antiparallel G-strands. This telomeric G-quadruplex structure contains three G-tetrads with mixed G-arrangements, which are connected consecutively with a double-chain-reversal side loop and two lateral loops, each consisting of three nucleotides TTA. This intramolecular hybrid-type telomeric G-quadruplex structure formed in K+ solution is distinct from those reported on the 22 nt Tel22 in Na+ solution and in crystalline state in the presence of K+, and appears to be the predominant conformation for the extended 26 nt telomeric sequence Tel26 in the presence of K+, regardless of the presence or absence of Na+. Furthermore, the addition of K+ readily converts the Na+-form conformation to the K+-form hybrid-type G-quadruplex. Our results explain all the reported experimental data on the human telomeric G-quadruplexes formed in the presence of K+, and provide important insights for understanding the polymorphism and interconversion of various G-quadruplex structures formed within the human telomeric sequence, as well as the effects of sequence and cations. This hybrid-type G-quadruplex topology suggests a straightforward pathway for the secondary structure formation with effective packing within the extended human telomeric DNA. The hybrid-type telomeric G-quadruplex is most likely to be of pharmacological relevance, and the distinct folding topology of this G-quadruplex suggests that it can be specifically targeted by G-quadruplex interactive small molecule drugs.

Dataset Information

Cutting antiparallel DNA strands in a single active site.

Publications

Cutting antiparallel DNA strands in a single active site.

Similar Datasets

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