Characterization of Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease, reveals a unique mode of DNA-binding, helical distortion and cleavage, compared to 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 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, Cu/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 possess 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 LAGLIDADG family of homing endonucleases

Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

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 ctive 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 active-site 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 PI-MleI, 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 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.

Structural and functional characteristics of homing endonucleases

Mobile genetic elements constitute a remarkably diverse group of nonessential selfish genes that provide no apparent function to the host. These selfish genes have been implicated in host extinction, speciation and architecture of genetic systems. Homing endonucleases, encoded by the open reading frames embedded in introns or inteins of mobile genetic elements, possess double-stranded DNA-specific endonuclease activity. They inflict sequence-specific double-strand breaks at or near the homing site in intron- or intein-less allele. Subsequently, through nonreciprocal exchange the insertion sequence (intron or intein) is transferred from an intein- or intron-containing allele to an intein- or intron-less allele. The components of host double-strand break repair pathway are thought to finish the "homing" process. Several lines of evidence suggest that homing endonucleases are capable of promoting transposition into ectopic sites within or across genomes for their survival as well as dispersal in natural populations. The occurrence of inteins at high frequencies serves as instructive models for understanding the mechanistic aspects of the process of homing and its evolution. This review focuses on genetic, biochemical, structural, and phylogenetic aspects of homing endonucleases, and their comparison with restriction endonucleases.

Homing abilities of the tropical primitively eusocial paper wasp Ropalidia marginata

Compared to our extensive knowledge about the navigation and homing abilities of ants and bees, we know rather little about these phenomena in social wasps. Here, we report the homing abilities of the tropical primitively eusocial wasp Ropalidia marginata and the factors that affect their homing success. To determine from how far these wasps can return to their nests, we transported foragers blindfold and released them at gradually increasing distances from their nests in four cardinal directions. Their homing success was determined by checking their presence on their nests on three consecutive nights. All foragers (56 individuals, 115 releases) returned back from an area of 0.73 +/- A 0.25 km(2) on the day of release (minimal homing area), whereas 83.8 % of the foragers (217 individuals, 420 releases) returned when we enlarged the area of release to 6.22 +/- A 0.66 km(2) around their nests (maximal homing area). Of 66 releases, no wasps returned from beyond the maximal homing area. The minimal homing area might be familiar to the foragers because they probably routinely forage in this area and the maximal homing area represents the maximum distances from which the wasps are capable of returning to their nests, with or without familiarity.

Computationally Efficient Sub-optimal Mid Course Guidance Using Model Predictive Static Programming (MPSP)

For a homing interceptor, suitable initial condition must be achieved by mid course guidance scheme for its maximum effectiveness. To achieve desired end goal of any mid course guidance scheme, two point boundary value problem must be solved online with all realistic constrain. A Newly developed computationally efficient technique named as MPSP (Model Predictive Static Programming) is utilized in this paper for obtaining suboptimal solution of optimal mid course guidance. Time to go uncertainty is avoided in this formulation by making use of desired position where midcourse guidance terminate and terminal guidance takes over. A suitable approach angle towards desired point also can be specified in this guidance law formulation. This feature makes this law particularly attractive because warhead effectiveness issue can be indirectly solved in mid course phase.