Mycobacterium tuberculosis RecA Intein Possesses a Novel ATP-dependent Site-specific Double-stranded DNA Endonuclease Activity

Guhan, N.; Muniyappa, K.

doi:10.1074/jbc.m112365200

Cited by 18 publications

(34 citation statements)

References 46 publications

(52 reference statements)

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“…The exact sequence of events that lead to recognition and cleavage of DNA by homing endonucleases is poorly understood. In addition, in no case the molecular mechanism underlying cleavage of ectopic DNA sites by an intein endonuclease has been elucidated.Mycobacterium tuberculosis RecA intein (PI-MtuI) 1 is a member of the LAGLIDADG superfamily of homing endonucleases (15)(16)(17)(18). In previous studies, we showed that PI-MtuI is a novel homing endonuclease, which inflicted a staggered doublestrand break 24 bp upstream of the intein insertion site in the inteinless recA allele (henceforth called cognate site) (18).…”

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“…In previous studies, we showed that PI-MtuI is a novel homing endonuclease, which inflicted a staggered doublestrand break 24 bp upstream of the intein insertion site in the inteinless recA allele (henceforth called cognate site) (18). Typically, Mg 2ϩ is the preferred metal ion and the only cofactor required for cleavage of inteinless alleles by the LAGLIDADG family of homing endonucleases.…”

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The RecA Intein of Mycobacterium tuberculosisPromotes Cleavage of Ectopic DNA Sites

Guhan

Muniyappa

2002

Journal of Biological Chemistry

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The RecA intein of Mycobacterium tuberculosis, a novel double-stranded DNA endonuclease, requires both Mn 2؉ and ATP for efficient cleavage of the inteinless recA allele. In this study, we show that Mg 2؉ alone was sufficient to stimulate PI-MtuI to cleave doublestranded DNA at ectopic sites. In the absence of Mg 2؉ , PI-MtuI formed complexes with topologically different forms of DNA containing ectopic recognition sequences with equal affinity but failed to cleave DNA. We observed that PI-MtuI was able to inflict double-strand breaks robustly within the ectopic recognition sequence to generate either a blunt end or 1-2-nucleotide 3-hydroxyl overhangs. Mobile inteins and introns are genetic elements capable of self-propagation by "homing" into host genes in a wide variety of organisms: eubacteria, eukarya, archaea, and viruses (reviewed in Ref. 1). The process is promoted by a homing endonuclease, which is encoded by an open reading frame embedded within the genetic element. The genes for homing endonucleases are found among group I and group II introns, archaeal introns, intein-coding sequences, and free standing open reading frames (reviewed in Refs. 1-6). Inteins are genetic elements present within protein-coding sequences with dual function: protein-splicing and homing endonuclease activities. They are believed to play a central role in rearrangement of organelle as well as nuclear genomes (1-6). The hallmark of homing endonucleases is their ability to recognize and cleave extended asymmetric sequences (14 -40 bp) that are generally centered on the intein insertion site in inteinless alleles (1, 7). Homing endonucleases can be classified into four families based on the presence of conserved motifs: LAGLIDADG, GIY-YIG, His-Cys box, and H-N-H (1,5,6,8). Among these, the LAGLIDADG family is the largest, widespread and much studied class of homing endonucleases. 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 (9 -12). Under standard assay conditions in vitro, these enzymes are extremely specific for their recognition sites. However, recent evidence suggests that self-splicing group II introns transpose into ectopic DNA sites that resemble their natural homing sites (13,14). The transposition of group II introns involves reverse splicing of the intron into the intronless allele, which are then reverse transcribed to give complementary DNA. Following the synthesis of the second strand, the intron is incorporated into the genomic DNA by homologous recombination (1-6). But equivalent information is not available for any intein endonuclease. The exact sequence of events that lead to recognition and cleavage of DNA by homing endonucleases is poorly understood. In addition, in no case the molecular mechanism underlying cleavage of ectopic DNA sites by an intein endonuclease has been elucidated.Mycobacterium tuberculosis RecA intein (PI-MtuI) 1 is a member of the LAGLIDADG...

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The RecA Intein of Mycobacterium tuberculosisPromotes Cleavage of Ectopic DNA Sites

Guhan

Muniyappa

2002

Journal of Biological Chemistry

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“…The K d values for PI-MleI and its variants are comparable to the K d values determined for the binding of other LAGLIDADG-type enzymes to their cognate DNA sequences. 12,36,37 The specificity of interaction of PI-MleI variants with target DNA sequence was determined using a salt-titration assay. The higher salt-titration midpoint value ($ 0.3 M), except in the case of D128A variant (Fig.…”

Section: Dna-binding Analysis Of Pi-mlei and Its Variants By Electropmentioning

confidence: 99%

“…[3][4][5] Several lines of evidence suggest that a variety of divalent cations can support cleavage reactions by HEases, but they do not influence DNA-binding. [10][11][12] Most HEases prefer oxophilic Mg 2þ , some use thiophilic Mn 2þ , whereas others can use both for optimal catalysis. 10,11 Cocrystal structures of several LAGLI-DADG endonucleases including I-CreI, 13 I-MsoI, 14 and I-SceI 15 indicated the presence of three divalent cations, coordinated by a pair of conserved aspartate residues at the active site.…”

Section: Introductionmentioning

confidence: 99%

“…23 In contrast, the sequences of recA intervening sequences are different in the two organisms. 23 Previously, we have shown that M. tuberculosis RecA intein (PIMtuI) recognizes a unique 45-bp asymmetrical DNA sequence, 12 a feature similar to that of many LAGLI-DADG-type HEases. 24 However, unlike other HEases, PI-MtuI possesses DNA-stimulated ATPase activity consistent with the presence of its ATP-binding motif.…”

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Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

2009

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Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their active center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the activesite residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp 122 (in Block C) and Asp 193 (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp 218 (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PIMleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp 122 and Additional Supporting Information may be found in the online version of this article.Abbreviations: 2-AP, 2 aminopurine; bp, base pair; BPB, bromophenol blue; EDTA, ethylene diamine tetraacetic acid; form I DNA, negatively supercoiled DNA; form II DNA, nicked circular DNA; form III DNA, linear double-stranded DNA; ODN, oligonucleotide; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PI-MleI, M. leprae RecA intein; SDS, sodium dodecyl sulfate. Asp 193 in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

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A survey of the year 2002 commercial optical biosensor literature

Rich

Myszka

2003

J of Molecular Recognition

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We have compiled 819 articles published in the year 2002 that involved commercial optical biosensor technology. The literature demonstrates that the technology's application continues to increase as biosensors are contributing to diverse scientific fields and are used to examine interactions ranging in size from small molecules to whole cells. Also, the variety of available commercial biosensor platforms is increasing and the expertise of users is improving. In this review, we use the literature to focus on the basic types of biosensor experiments, including kinetics, equilibrium analysis, solution competition, active concentration determination and screening. In addition, using examples of particularly well-performed analyses, we illustrate the high information content available in the primary response data and emphasize the impact of including figures in publications to support the results of biosensor analyses.

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Mycobacterium tuberculosis RecA Intein Possesses a Novel ATP-dependent Site-specific Double-stranded DNA Endonuclease Activity

Cited by 18 publications

References 46 publications

The RecA Intein of Mycobacterium tuberculosisPromotes Cleavage of Ectopic DNA Sites

The RecA Intein of Mycobacterium tuberculosisPromotes Cleavage of Ectopic DNA Sites

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not

A survey of the year 2002 commercial optical biosensor literature

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Mycobacterium tuberculosis RecA Intein Possesses a Novel ATP-dependent Site-specific Double-stranded DNA Endonuclease Activity

Cited by 18 publications

References 46 publications

The RecA Intein of Mycobacterium tuberculosisPromotes Cleavage of Ectopic DNA Sites

The RecA Intein of Mycobacterium tuberculosisPromotes Cleavage of Ectopic DNA Sites

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

A survey of the year 2002 commercial optical biosensor literature

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Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp¹²² and Asp¹⁹³ are crucial to the double‐stranded DNA cleavage activity whereas Asp²¹⁸ is not