Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.
Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.
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DOI：
10.1093/nar/gkv521
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发表时间：
2015-07-13
影响因子：
14.9
通讯作者：
Stark WM
中科院分区：
文献类型：
--
作者：
Olorunniji FJ;McPherson AL;Pavlou HJ;McIlwraith MJ;Brazier JA;Cosstick R;Stark WM
为了分析丝氨酸重组酶 Tn3 解离酶催化的 DNA 链裂解的机制和动力学，我们在两条 DNA 链之一中制作了带有单链切口的修饰重组位点。作用于这些位点的解离酶非常快速地切割完整链，产生异常的半位点产物并积累。我们建议这些反应模拟未修饰位点的第二链裂解。切割发生在两个位点的突触中，由解离酶四聚体连接在一起；一个位点的裂解会刺激伴侣位点的裂解。切口位点底物裂解后，未与解离酶亚基共价连接的半位点迅速从突触上解离，从而使整个复合物不稳定。因此，自然反应中间体中的共价解离酶-DNA 连接发挥着重要的 DNA 束缚功能。对可裂解磷酸二酯位置处的切口位点底物进行化学修饰，可消除或抑制拆分酶介导的裂解，并对拆分酶结合和突触产生影响，从而深入了解丝氨酸重组酶催化机制以及拆分酶如何与底物 DNA 相互作用。
To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase–DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA.
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影响因子：
14.9
作者：
Olorunniji FJ;He J;Wenwieser SV;Boocock MR;Stark WM
通讯作者：
Stark WM
影响因子：
64.5
作者：
NUNESDUBY, SE;MATSUMOTO, L;LANDY, A
通讯作者：
LANDY, A
影响因子：
14.9
作者：
BURGIN, AB;HUIZENGA, BN;NASH, HA
通讯作者：
NASH, HA
影响因子：
5.6
作者：
Blakely, GW;Davidson, AO;Sherratt, DJ
通讯作者：
Sherratt, DJ
影响因子：
14.8
作者：
Gaynor, James W.;Bentley, Joanne;Cosstick, Richard
通讯作者：
Cosstick, Richard