Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile.
Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile.
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DOI：
10.1098/rsob.160272
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发表时间：
2016-12
期刊：
影响因子：
5.8
通讯作者：
Smits WK
中科院分区：
文献类型：
--
作者：
van Eijk E;Paschalis V;Green M;Friggen AH;Larson MA;Spriggs K;Briggs GS;Soultanas P;Smits WK
DNA复制在生命的所有领域都是一个必要的和保守的过程，可以作为开发新的抗菌剂的目标。然而，这种发展受到微妙的机制差异和对致病微生物DNA复制的有限理解的阻碍。艰难梭菌是卫生保健相关腹泻的主要原因，其DNA复制机制几乎没有特征。我们鉴定并表征了难辨梭菌的复制解旋酶（CD3657）、解旋酶装载器atp酶（CD3654）和引物酶（CD1454）的机制细节，并在体外重建了解旋酶和引物酶的活性。我们证明了解旋酶装载器和解旋酶之间的直接和依赖于atp的相互作用。此外，我们发现解旋酶的活性依赖于体外引物酶的存在。引物酶固有的三核苷酸特异性是由一个赖氨酸残基决定的，与极端嗜热菌水蛭的引物酶相似。然而，解旋酶的存在使得从非优选的三核苷酸中更有效地重新合成RNA引物。因此，在所有生物体的DNA复制过程中，装载物-解旋酶-引发酶的相互作用至关重要地介导解旋酶的装载和激活，艰难梭菌与经过充分研究的革兰氏阳性枯草芽孢杆菌模型的相互作用截然不同。
DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. However, such developments are hindered by subtle mechanistic differences and limited understanding of DNA replication in pathogenic microorganisms. Clostridium difficile is the main cause of healthcare-associated diarrhoea and its DNA replication machinery is virtually uncharacterized. We identify and characterize the mechanistic details of the putative replicative helicase (CD3657), helicase-loader ATPase (CD3654) and primase (CD1454) of C. difficile, and reconstitute helicase and primase activities in vitro. We demonstrate a direct and ATP-dependent interaction between the helicase loader and the helicase. Furthermore, we find that helicase activity is dependent on the presence of primase in vitro. The inherent trinucleotide specificity of primase is determined by a single lysine residue and is similar to the primase of the extreme thermophile Aquifex aeolicus. However, the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, loader–helicase–primase interactions, which crucially mediate helicase loading and activation during DNA replication in all organisms, differ critically in C. difficile from that of the well-studied Gram-positive Bacillus subtilis model.
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通过细菌启动器拉伸的DNA促进了复制起源。
影响因子：
64.8
作者：
Duderstadt, Karl E.;Chuang, Kevin;Berger, James M.
通讯作者：
Berger, James M.
影响因子：
3.6
作者：
Bruand, C;Velten, M;Polard, P
通讯作者：
Polard, P
影响因子：
2.9
作者：
Biswas, Subhasis B.;Biswas-Fiss, Esther E.
通讯作者：
Biswas-Fiss, Esther E.
影响因子：
14.9
作者：
Bazin A;Cherrier MV;Gutsche I;Timmins J;Terradot L
通讯作者：
Terradot L
影响因子：
6.4
作者：
Dembek M;Barquist L;Boinett CJ;Cain AK;Mayho M;Lawley TD;Fairweather NF;Fagan RP
通讯作者：
Fagan RP