Plasmid Replication Initiator RepB Forms a Hexamer Reminiscent of Ring Helicases and Has Mobile Nuclease Domains

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 2009 May 15

PMID 19440202

Citations 31

Authors

D Roeland Boer

Jose A Ruiz-Maso

Jose R Lopez-Blanco

Alexander G Blanco

Mireia Vives-Llacer

Pablo Chacon

Isabel Uson

F Xavier Gomis-Ruth

Manuel Espinosa

Oscar Llorca

Gloria Del Solar

Miquel Coll

Affiliations

Soon will be listed here.

Abstract

RepB initiates plasmid rolling-circle replication by binding to a triple 11-bp direct repeat (bind locus) and cleaving the DNA at a specific distant site located in a hairpin loop within the nic locus of the origin. The structure of native full-length RepB reveals a hexameric ring molecule, where each protomer has two domains. The origin-binding and catalytic domains show a three-layer alpha-beta-alpha sandwich fold. The active site is positioned at one of the faces of the beta-sheet and coordinates a Mn2+ ion at short distance from the essential nucleophilic Y99. The oligomerization domains (ODs), each consisting of four alpha-helices, together define a compact ring with a central channel, a feature found in ring helicases. The toroidal arrangement of RepB suggests that, similar to ring helicases, it encircles one of the DNA strands during replication to confer processivity to the replisome complex. The catalytic domains appear to be highly mobile with respect to ODs. This mobility may account for the adaptation of the protein to two distinct DNA recognition sites.

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References

Pettersen E, Goddard T, Huang C, Couch G, Greenblatt D, Meng E . UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13):1605-12. DOI: 10.1002/jcc.20084. View

Murshudov G, Vagin A, Dodson E . Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997; 53(Pt 3):240-55. DOI: 10.1107/S0907444996012255. View

Ruiz-Maso J, Lopez-Zumel C, Menendez M, Espinosa M, Del Solar G . Structural features of the initiator of replication protein RepB encoded by the promiscuous plasmid pMV158. Biochim Biophys Acta. 2004; 1696(1):113-9. DOI: 10.1016/j.bbapap.2003.09.010. View

Ruiz-Maso J, Lurz R, Espinosa M, Del Solar G . Interactions between the RepB initiator protein of plasmid pMV158 and two distant DNA regions within the origin of replication. Nucleic Acids Res. 2007; 35(4):1230-44. PMC: 1851628. DOI: 10.1093/nar/gkl1099. View

Espinosa M, Lopez P, Lacks S . Transfer and expression of recombinant plasmids carrying pneumococcal mal genes in Bacillus subtilis. Gene. 1984; 28(3):301-10. DOI: 10.1016/0378-1119(84)90147-1. View

Campos-Olivas R, Louis J, Clerot D, Gronenborn B, Gronenborn A . The structure of a replication initiator unites diverse aspects of nucleic acid metabolism. Proc Natl Acad Sci U S A. 2002; 99(16):10310-5. PMC: 124910. DOI: 10.1073/pnas.152342699. View

Petit M, Dervyn E, Rose M, Entian K, McGovern S, Ehrlich S . PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling-circle replication. Mol Microbiol. 1998; 29(1):261-73. DOI: 10.1046/j.1365-2958.1998.00927.x. View

Kawabata T . MATRAS: A program for protein 3D structure comparison. Nucleic Acids Res. 2003; 31(13):3367-9. PMC: 168987. DOI: 10.1093/nar/gkg581. View

McCoy A, Grosse-Kunstleve R, Storoni L, Read R . Likelihood-enhanced fast translation functions. Acta Crystallogr D Biol Crystallogr. 2005; 61(Pt 4):458-64. DOI: 10.1107/S0907444905001617. View

10.

. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr. 1994; 50(Pt 5):760-3. DOI: 10.1107/S0907444994003112. View

11.

Zhao A, Ansari R, Schmidt M, Khan S . An oligonucleotide inhibits oligomerization of a rolling circle initiator protein at the pT181 origin of replication. J Biol Chem. 1998; 273(26):16082-9. DOI: 10.1074/jbc.273.26.16082. View

12.

Navaza J . Implementation of molecular replacement in AMoRe. Acta Crystallogr D Biol Crystallogr. 2001; 57(Pt 10):1367-72. DOI: 10.1107/s0907444901012422. View

13.

Vega-Rocha S, Byeon I, Gronenborn B, Gronenborn A, Campos-Olivas R . Solution structure, divalent metal and DNA binding of the endonuclease domain from the replication initiation protein from porcine circovirus 2. J Mol Biol. 2007; 367(2):473-87. DOI: 10.1016/j.jmb.2007.01.002. View

14.

McRee D . XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. J Struct Biol. 1999; 125(2-3):156-65. DOI: 10.1006/jsbi.1999.4094. View

15.

Bruand C, Ehrlich S . UvrD-dependent replication of rolling-circle plasmids in Escherichia coli. Mol Microbiol. 2000; 35(1):204-10. DOI: 10.1046/j.1365-2958.2000.01700.x. View

16.

Sorzano C, Marabini R, Velazquez-Muriel J, Bilbao-Castro J, Scheres S, Carazo J . XMIPP: a new generation of an open-source image processing package for electron microscopy. J Struct Biol. 2004; 148(2):194-204. DOI: 10.1016/j.jsb.2004.06.006. View

17.

Naqvi A, Anand S, Kramer M, Munshi R, Khan S . Biochemical characterization of the Staphylococcus aureus PcrA helicase and its role in plasmid rolling circle replication. J Biol Chem. 2002; 277(48):45880-6. DOI: 10.1074/jbc.M207383200. View

18.

Moscoso M, Del Solar G, Espinosa M . Specific nicking-closing activity of the initiator of replication protein RepB of plasmid pMV158 on supercoiled or single-stranded DNA. J Biol Chem. 1995; 270(8):3772-9. DOI: 10.1074/jbc.270.8.3772. View

19.

Scheres S, Valle M, Nunez R, S Sorzano C, Marabini R, Herman G . Maximum-likelihood multi-reference refinement for electron microscopy images. J Mol Biol. 2005; 348(1):139-49. DOI: 10.1016/j.jmb.2005.02.031. View

20.

Ludtke S, Baldwin P, Chiu W . EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol. 1999; 128(1):82-97. DOI: 10.1006/jsbi.1999.4174. View