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Inference of relationships in the 'twilight zone' of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII.

ABSTRACT: Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insignificant, unless compared among proteins that recognize identical or very similar sequences. Bsp6I is a Type II REase, which recognizes the palindromic DNA sequence 5'GCNGC and cleaves between the cytosine and the unspecified nucleotide in both strands, generating a double-strand break with 5'-protruding single nucleotides. There are no solved structures of REases that recognize similar DNA targets or generate cleavage products with similar characteristics. In straightforward comparisons, the Bsp6I sequence shows no significant similarity to REases with known structures. However, using a fold-recognition approach, we have identified a remote relationship between Bsp6I and the structure of PvuII. Starting from the sequence-structure alignment between Bsp6I and PvuII, we constructed a homology model of Bsp6I and used it to predict functionally significant regions in Bsp6I. The homology model was supported by site-directed mutagenesis of residues predicted to be important for dimerization, DNA binding and catalysis. Completing the picture of sequence-structure-function relationships in protein superfamilies becomes an essential task in the age of structural genomics and our study may serve as a paradigm for future analyses of superfamilies comprising strongly diverged members with little or no sequence similarity.

SUBMITTER: Pawlak SD

PROVIDER: S-EPMC548357 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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Publications

Inference of relationships in the 'twilight zone' of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII.

Pawlak Sebastian D SD Radlinska Monika M Chmiel Agnieszka A AA Bujnicki Janusz M JM Skowronek Krzysztof J KJ

Nucleic acids research 20050131 2

Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insignificant, unless compared among proteins that recognize identical or very similar sequences. Bsp6I is a Type II REase, which recognizes the palindromic DNA sequence 5'GCNGC and cleaves between the cyto ...[more]

PMID: 15684412

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Project description:What happens in the brain when conscious awareness of the surrounding world fades? We manipulated consciousness in two experiments in a group of healthy males and measured brain activity with positron emission tomography. Measurements were made during wakefulness, escalating and constant levels of two anesthetic agents (experiment 1, n = 39), and during sleep-deprived wakefulness and non-rapid eye movement sleep (experiment 2, n = 37). In experiment 1, the subjects were randomized to receive either propofol or dexmedetomidine until unresponsiveness. In both experiments, forced awakenings were applied to achieve rapid recovery from an unresponsive to a responsive state, followed by immediate and detailed interviews of subjective experiences during the preceding unresponsive condition. Unresponsiveness rarely denoted unconsciousness, as the majority of the subjects had internally generated experiences. Unresponsive anesthetic states and verified sleep stages, where a subsequent report of mental content included no signs of awareness of the surrounding world, indicated a disconnected state. Functional brain imaging comparing responsive and connected versus unresponsive and disconnected states of consciousness during constant anesthetic exposure revealed that activity of the thalamus, cingulate cortices, and angular gyri are fundamental for human consciousness. These brain structures were affected independent from the pharmacologic agent, drug concentration, and direction of change in the state of consciousness. Analogous findings were obtained when consciousness was regulated by physiological sleep. State-specific findings were distinct and separable from the overall effects of the interventions, which included widespread depression of brain activity across cortical areas. These findings identify a central core brain network critical for human consciousness.SIGNIFICANCE STATEMENT Trying to understand the biological basis of human consciousness is currently one of the greatest challenges of neuroscience. While the loss and return of consciousness regulated by anesthetic drugs and physiological sleep are used as model systems in experimental studies on consciousness, previous research results have been confounded by drug effects, by confusing behavioral "unresponsiveness" and internally generated consciousness, and by comparing brain activity levels across states that differ in several other respects than only consciousness. Here, we present carefully designed studies that overcome many previous confounders and for the first time reveal the neural mechanisms underlying human consciousness and its disconnection from behavioral responsiveness, both during anesthesia and during normal sleep, and in the same study subjects.

Dataset Information

Inference of relationships in the 'twilight zone' of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII.

Publications

Inference of relationships in the 'twilight zone' of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII.

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