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J Bitinaite

Publications and source records attributed to J Bitinaite.

8 recordsLinked to original sources
PubMed1999

Genetic analysis of the base-specific contacts of BamHI restriction endonuclease.

Here, we investigate the highly specific interaction of the BamHI endonuclease with its cognate recognition sequence GGATCC by determining which amino acid residues can be substituted at the DNA interface while maintaining specificity. Mutational studies, together with the structural determination of the restriction endonuclease BamHI have revealed the amino acid residues which are involved in DNA catalysis and those which play a role in the specific binding of the enzyme to its cognate DNA recognition sequence. Amino acid residues N116, S118, R122, D154 and R155 are involved in DNA sequence recognition and are located in the major groove in close proximity to the nucleotide bases comprising the recognition sequence. Cassette mutagenesis of these amino acids, together with in vivo transcriptional interference selection, was used to identify an array of substitutions which maintain site-specific binding to the cognate GGATCC sequence. This approach has demonstrated the extent of acceptable variation among amino acid residues which are directly involved in site-specific binding. One variant, double mutant N116H, S118G was found to cleave DNA only when the adenine base in the recognition site is methylated.

Amino Acid Sequence↗
PubMed1998

Structure of FokI has implications for DNA cleavage.

FokI is a member an unusual class of restriction enzymes that recognize a specific DNA sequence and cleave nonspecifically a short distance away from that sequence. FokI consists of an N-terminal DNA recognition domain and a C-terminal cleavage domain. The bipartite nature of FokI has led to the development of artificial enzymes with novel specificities. We have solved the structure of FokI to 2.3 A resolution. The structure reveals a dimer, in which the dimerization interface is mediated by the cleavage domain. Each monomer has an overall conformation similar to that found in the FokI-DNA complex, with the cleavage domain packing alongside the DNA recognition domain. In corroboration with the cleavage data presented in the accompanying paper in this issue of Proceedings, we propose a model for FokI DNA cleavage that requires the dimerization of FokI on DNA to cleave both DNA strands.

Amino Acid Sequence↗
PubMed1998

FokI dimerization is required for DNA cleavage.

FokI is a type IIs restriction endonuclease comprised of a DNA recognition domain and a catalytic domain. The structural similarity of the FokI catalytic domain to the type II restriction endonuclease BamHI monomer suggested that the FokI catalytic domains may dimerize. In addition, the FokI structure, presented in an accompanying paper in this issue of Proceedings, reveals a dimerization interface between catalytic domains. We provide evidence here that FokI catalytic domain must dimerize for DNA cleavage to occur. First, we show that the rate of DNA cleavage catalyzed by various concentrations of FokI are not directly proportional to the protein concentration, suggesting a cooperative effect for DNA cleavage. Second, we constructed a FokI variant, FokN13Y, which is unable to bind the FokI recognition sequence but when mixed with wild-type FokI increases the rate of DNA cleavage. Additionally, the FokI catalytic domain that lacks the DNA binding domain was shown to increase the rate of wild-type FokI cleavage of DNA. We also constructed an FokI variant, FokD483A, R487A, which should be defective for dimerization because the altered residues reside at the putative dimerization interface. Consistent with the FokI dimerization model, the variant FokD483A, R487A revealed greatly impaired DNA cleavage. Based on our work and previous reports, we discuss a pathway of DNA binding, dimerization, and cleavage by FokI endonuclease.

Base Sequence↗
PubMed1992

Alw26I, Eco31I and Esp3I--type IIs methyltransferases modifying cytosine and adenine in complementary strands of the target DNA.

The specificity of three DNA methyltransferases M.Alw26I, M.Eco31I and M.Esp3I, isolated from Acinetobacter Iwoffi RFL26, Escherichia coli RFL31 and Hafnia alvei RFL3+, respectively, was determined. All the enzymes methylate both strands of asymmetric recognition sites yielding m5C in the top-strand and m6A in the bottom-strand, as below: 5'-GTm5CTC 5'-GGTm5CTC 5'-CGTm5CTC 3'-Cm6AGAG 3'-CCm6AGAG 3'-GCm6AGAG (M.Alw26I) (M.Eco31I) (M.Esp3I) They are the first members of type IIs methyltransferases that modify different types of nucleotides in the recognition sequence.

Acinetobacter↗