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*
AUTOR(ES)
Singh, Pawan
FONTE
American Society for Biochemistry and Molecular Biology
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2757992Documentos Relacionados
- Mycobacterium tuberculosis RecA intein, a LAGLIDADG homing endonuclease, displays Mn2+ and DNA-dependent ATPase activity
- In Vivo Splicing and Functional Characterization of Mycobacterium leprae RecA
- Binding, bending and cleavage of DNA substrates by the homing endonuclease Pl-SceI.
- The spread of LAGLIDADG homing endonuclease genes in rDNA
- Characterization of the Two Mycobacterium tuberculosis recA Promoters