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Mycobacterium tuberculosis RecA intein, a novel LAGLIDADG homing endonuclease, displays dual target specificity in the presence of alternative cofactors

Electronic Theses of Indian Institute of Science

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Title Mycobacterium tuberculosis RecA intein, a novel LAGLIDADG homing endonuclease, displays dual target specificity in the presence of alternative cofactors
 
Creator Guhan, N
 
Subject Biochemistry
Mycobacterium tuberculosis
LAGLIDADG
DNA
Mycobacteria
 
Description Mobile inteins and introns are genetic elements capable of self-propagation by “homing” into host genes and occur in entire taxonomy: eubacteria, eukarya, archaea and viruses. The process of “homing” is promoted by an endonuclease encoded by the open reading frame (ORF) embedded within the genetic element. Homing endonucleases are encoded by group I and group II introns, archaeal introns, inteins, and free standing ORFs. They are believed to play a central role in rearrangement of organelle as well as nuclear genomes. Inteins are genetic elements present within protein-coding genes with dual function: protein-splicing and homing endonuclease activities. One hallmark of homing endonucleases is their ability to recognize and cleave extended degenerate asymmetric sequences (14 - 40 bp) in intein- or intron-less alleles. Homing endonucleases are classified into four families based on the presence of LAGLIDADG, GIY-YIG, His-Cys box, or H-N-H conserved motifs. Among these, LAGLIDADG family is the largest, widespread and well-studied class. Structural and biochemical studies have demonstrated that homing endonucleases with one LAGLIDADG motif act as homodimers, whereas enzymes with two such motifs function as monomers during catalysis. In vitro, these enzymes are extremely specific for their recognition sites and they prefer Mg2+ as the metal-ion cofactor.

Unlike Escherichia coli, recA of Mycobacterium tuberculosis and Mycobacterium leprae contain in-frame insertion of an intein-coding sequence. In addition to recA,scrutiny of M. tuberculosis genome revealed that intein-coding sequences are present in the ORFs of dnaA and Rv1461 (pps1). M. tuberculosis recA encodes a 85 kDa precursor
protein. Amino acid sequence comparison between M. tuberculosis RecA precursor and the prototype E. coli RecA displayed high degree of homology at the amino-terminal (1 -254 amino acid residues) and carboxyl-terminal (694 - 790 amino acid residues)domains of the RecA precursor. The central domain comprising 440 amino acid residues
showed significant homology to the members of the LAGLIDADG super-family of intein homing endonucleases. Following the synthesis of precursor protein, RecA intein and active RecA are generated by protein splicing reaction. The protein splicing reaction of RecA intein has been studied extensively; however, its endonuclease activity remained obscure.

To identify the biochemical function of M. tuberculosis RecA intein (PI-MtuI), the
intervening sequence from recA was cloned, overexpressed in E. coli and purified to
homogeneity. The identity of PI-MtuI was ascertained by sequencing 10 amino acid
residues at the amino-terminal end and by Western blot analysis using polyclonal antibodies raised against precursor RecA.
 
Publisher Indian Institute of Science
 
Contributor Muniyappa, K
 
Date 2005-05-10T05:26:43Z
2005-05-10T05:26:43Z
2005-05-10T05:26:43Z
2002-12
 
Type Electronic Thesis and Dissertation
 
Format 8350616 bytes
application/pdf
 
Identifier http://etd.iisc.ernet.in/handle/2005/117
111196387
 
Language en
 
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