Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases

Francia M.V.; Clewell D.B.; De La Cruz F.Moncalián G.

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Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases

机译：Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases

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摘要

Plasmid pAD1 is a 60-kb conjugative element commonly found in clinical isolates of Enterococcus faecalis. The relaxase TraX and the primary origin of transfer oriT2 are located close to each other and have been shown to be essential for conjugation. The oriT2 site contains a large inverted repeat (where the nic site is located) adjacent to a series of short direct repeats. TraX does not show any of the typical relaxase sequence motifs but is the prototype of a unique family of relaxases (MOBC). The present study focuses on the genetic, biochemical, and structural analysis of TraX, whose 3D structure could be predicted by protein threading. The structure consists of two domains: (i) an N-terminal domain sharing the topology of the DNA binding domain of the MarR family of transcriptional regulators and (ii) a C-terminal catalytic domain related to the PD-(D/E)XK family of restriction endonucleases. Alignment of MOBC relaxase amino acid sequences pointed to several conserved polar amino acid residues (E28, D152, E170, E172, K176, R180, Y181, and Y203) that were mutated to alanine. Functional analysis of these mutants (in vivo DNA transfer and cleavage assays) revealed the importance of these residues for relaxase activity and suggests Y181 as a potential catalytic residue similarly to His-hydrophobe-His relaxases. We also show that TraX binds specifically to dsDNA containing the oriT2 direct repeat sequences, confirming their role in transfer specificity. The results provide insights into the catalytic mechanism of MOBC relaxases, which differs radically from that of His-hydrophobe-His relaxases.

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《Proceedings of the National Academy of Sciences of the United States of America》 |2013年第33期|13606-13611|共6页
作者
Francia M.V.; Clewell D.B.; De La Cruz F.Moncalián G.;
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作者单位

Servicio de Microbiología, Hospital Universitario Marqués de Valdecilla, Instituto de Formación e Investigación Marqués de Valdecilla, Santander 39008, Spain;

Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, MI 48109, United States;

Departamento de Biología Molecular, Instituto de Biomedicina y Biotecnología de Cantabria, Universidad de Cantabria-Consejo Superior de Investigaciones Científicas-Sociedad, Santander 39011, Spain;

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收录信息美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
原文格式 PDF
正文语种英语
中图分类自然科学总论;
关键词
Catalytic domain; relaxases related; restriction endonucleases;

Catalytic domain of plasmid pAD1 relaxase TraX defines a group of relaxases related to restriction endonucleases

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