Characterization of Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease, reveals a unique mode of DNA binding, helical distortion, and cleavage compared with a canonical LAGLIDADG homing endonuclease.
Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of the M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches, including four complementary footprinting assays such as DNase I, copper-phenanthroline, methylation protection, and KMnO4, enhancement of 2-aminopurine fluorescence, and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site, and generates two staggered double strand breaks. Taken together, these results implicate that PI-MleI possesses a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of the LAGLIDADG family of homing endonucleases.
Singh
Pawan
P
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Tripathi
Pankaj
P
Silva
George H
GH
Pingoud
Alfred
A
Muniyappa
K
K
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D047668:Inteins / Q000502:physiology*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D009166:Mycobacterium leprae / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008345:Manganese; D011938:Rec A Recombinases; D004720:Endonucleases; D008274:Magnesium