Functional domains in protein TrwC of plasmid R388: dissected DNA strand transferase and DNA helicase activities reconstitute protein function

Journal of Molecular Biology
M LlosaFernando de la Cruz

Abstract

TrwC is a bifunctional enzyme that displays two biochemical activities essential for plasmid R388 conjugation: oriT-specific DNA strand-transferase and DNA helicase activities. We overproduced and purified different segments of the protein allowing us to map the relaxase and DNA helicase activities to separate regions of the protein. A peptide comprising the N-terminal 275 amino acid residues of the protein was able to catalyze DNA cleavage and strand-transfer reactions when using oligonucleotides encompassing the nic site, although a longer fragment of TrwC (348 amino acid residues) was required to produce the nick on a supercoiled double-stranded DNA substrate. The segment of the protein between amino acid residues 192 and 966 contained the ATPase and DNA helicase activities, while a peptide consisting of amino acid residues 346 to 966 lost both activities. The dimerization region lay in the 495 C-terminal amino acid residues. Two peptides containing the DNA strand-transferase and DNA helicase activities, respectively, could functionally substitute for TrwC in R388 conjugation although at a 10,000-fold lower efficiency. Thus, integrity of the covalent structure of the protein was required for efficient DNA transfer. It can be...Continue Reading

Citations

Oct 12, 2010·Biomolecular NMR Assignments·Nathan T WrightJoel F Schildbach
Jul 9, 2002·Molecular Microbiology·Matxalen LlosaFernando de la Cruz Fd
Jan 22, 2003·Environmental Microbiology·Alicia GreatedChristopher M Thomas
May 8, 2010·Nucleic Acids Research·Rebekah Potts NashMatthew R Redinbo
Aug 23, 2006·Journal of Bacteriology·Rembrandt J F HaftBeth Traxler
Aug 19, 2006·Journal of Bacteriology·Daniel Pérez-MendozaJuan Sanjuán
Sep 22, 2009·Journal of Bacteriology·Sanja MihajlovicEllen L Zechner
Sep 22, 2009·Journal of Bacteriology·Marta V SutEllen L Zechner
Oct 9, 2007·Journal of Bacteriology·Carolina Elvira César, Matxalen Llosa
Apr 30, 2013·Journal of Bacteriology·Cris Fernández-LópezD Roeland Boer
Jul 3, 2003·Journal of Bacteriology·José-Luis R ArrondoFélix M Goñi
May 20, 2004·Journal of Bacteriology·Matthew C A Smith, Christopher D Thomas
Jan 5, 2005·Journal of Bacteriology·Steven W Matson, Heather Ragonese
Sep 25, 2003·Proceedings of the National Academy of Sciences of the United States of America·Matthew J Harley, Joel F Schildbach
Aug 20, 2003·Proceedings of the National Academy of Sciences of the United States of America·Matxalen LlosaFernando de la Cruz
Nov 2, 2005·Proceedings of the National Academy of Sciences of the United States of America·Olga DraperMatxalen Llosa
Apr 21, 1999·Molecular Microbiology·G MoncaliánF de la Cruz
Oct 10, 2006·FEMS Microbiology Reviews·Raúl Fernández-LópezFernando de la Cruz
May 21, 2016·Frontiers in Molecular Biosciences·Esther Fernández-GonzálezMatxalen Llosa
Jul 31, 2007·The EMBO Journal·Blanca Gonzalez-PerezGabriel Moncalián
Nov 20, 2003·Nature Structural Biology·Alicia GuaschMiquel Coll
Dec 14, 2006·Molecular Microbiology·M Pilar Garcillán-BarciaFernando de la Cruz
Sep 25, 2003·The Journal of Biological Chemistry·Vanessa C Csitkovits, Ellen L Zechner
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Jul 11, 1997·Journal of Molecular Biology·G MoncaliánF de la Cruz
Jan 13, 2018·Microbiology Spectrum·María Getino, Fernando de la Cruz

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