Identification of amino acid residues critical for catalysis of holliday junction resolution by Mycoplasma genitalium RecU

Marcel Sluijter, Mohammad Aslam, Nico G. Hartwig, Annemarie M.C. van Rossum, Cornelis Vink*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

7 Citations (Scopus)

Abstract

The RecU protein from Mycoplasma genitalium, RecU Mge, is a 19.4-kDa Holliday junction (HJ) resolvase that binds in a nonspecific fashion to HJ substrates and, in the presence of Mn 2+, cleaves these substrates at a specific sequence (5'-G/TC2C/TTA/GG-3'). To identify amino acid residues that are crucial for HJ binding and/or cleavage, we generated a series of 16 deletion mutants (9 N- and 7 C-terminal deletion mutants) and 31 point mutants of RecU Mge. The point mutations were introduced at amino acid positions that are highly conserved among bacterial RecU-like sequences. All mutants were purified and tested for the ability to bind to, and cleave, HJ substrates. We found the five N-terminal and three C-terminal amino acid residues of RecU Mge to be dispensable for its catalytic activities. Among the 31 point mutants, 7 mutants were found to be inactive in both HJ binding and cleavage. Interestingly, in 12 other mutants, these two activities were uncoupled; while these proteins displayed HJ-binding characteristics similar to those of wild-type RecU Mge, they were unable to cleave HJ substrates. Thus, 12 amino acid residues were identified (E11, K31, D57, Y58, Y66, D68, E70, K72, T74, K76, Q88, and L92) that may play either a direct or indirect role in the catalysis of HJ resolution.

Original languageEnglish
Pages (from-to)3941-3948
Number of pages8
JournalJournal of Bacteriology
Volume193
Issue number15
DOIs
Publication statusPublished - Aug 2011

Fingerprint

Dive into the research topics of 'Identification of amino acid residues critical for catalysis of holliday junction resolution by Mycoplasma genitalium RecU'. Together they form a unique fingerprint.

Cite this