Conserved amino acid motifs from the novel Piv/MooV family of transposases and site-specific recombinases are required for catalysis of DNA inversion by Piv.
Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.
Tobiason
D M
DM
Department of Microbiology, University of Georgia, 527 Biological Sciences Bldg., Cedar Street, Athens, GA 30602-2605, USA.
Buchner
J M
JM
Thiel
W H
WH
Gernert
K M
KM
Karls
A C
AC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D007446:Chromosome Inversion; D017124:Conserved Sequence; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism*; D004251:DNA Transposable Elements / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D019426:Integrases; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009016:Moraxella / Q000201:enzymology / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D045522:Recombinases; D019895:Transposases / Q000737:chemistry* / Q000378:metabolism
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D045522:Recombinases; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D019895:Transposases; C115006:integron integrase IntI1