ABSTRACT
One hundred and forty isolates of thermophilic bacteria from the genus Thermus were screened for the presence of restriction endonuclease activity. Thermostable isoschizomers of restriction endonucleases, such as AceIII, BbvI, BglI, BsePI, FnuDII, HgiAI, MaeII, MboI, MseI, PvuII, StuI, TaqI, Tsp4CI, TspEI, XhoI and XmaIII, were isolated. Two restriction enzymes, TatI and TauI, recognizing novel degenerate sequences 5'-W (downward arrow)GTACW-3' and 5'-GCSG (downward arrow)C-3' respectively were partially purified and the recognition and cleavage sites were determined.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/metabolism , Thermus/enzymology , Base Sequence , DNA/chemistry , DNA/metabolism , Enzyme Stability , Hot Temperature , Substrate SpecificityABSTRACT
Methyltransferases (MTases) of procaryotes affect general cellular processes such as mismatch repair, regulation of transcription, replication, and transposition, and in some cases may be essential for viability. As components of restriction-modification systems, they contribute to bacterial genetic diversity. The genome of Helicobacter pylori strain 26695 contains 25 open reading frames encoding putative DNA MTases. To assess which MTase genes are active, strain 26695 genomic DNA was tested for cleavage by 147 restriction endonucleases; 24 were found that did not cleave this DNA. The specificities of 11 expressed MTases and the genes encoding them were identified from this restriction data, combined with the known sensitivities of restriction endonucleases to specific DNA modification, homology searches, gene cloning and genomic mapping of the methylated bases m(4)C, m(5)C, and m(6)A.
Subject(s)
DNA Modification Methylases/metabolism , Helicobacter pylori/enzymology , Cloning, Molecular , DNA Restriction Enzymes/metabolism , DNA, Bacterial/metabolism , Genes, Bacterial , Methyltransferases/genetics , Methyltransferases/metabolism , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
A new type II restriction endonuclease designated FspAI has been partially purified from a Flexibacter species Tv-m21K. FspAI recognizes the octanucleotide sequence 5'-RTGC/GCAY-3' and cleaves it in the center generating blunt-ended DNA fragments.
Subject(s)
Deoxyribonucleases, Type II Site-Specific/metabolism , Oligonucleotides/metabolism , Bacteria/enzymology , Base Sequence , Binding Sites/genetics , DNA/genetics , DNA/metabolism , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Oligonucleotides/genetics , Substrate SpecificityABSTRACT
A new restriction endonuclease Abe I has beenisolated from Azotobacter beijerinckii. This enzymerecognizes the asymmetric heptanucleotide sequence 5'-CCTCAGC-3' and cleaves within it symmetrically at positions -5/-2 in the opposing strands, producing three base protruding 5'-ends.
Subject(s)
Azotobacter/enzymology , Deoxyribonucleases, Type II Site-Specific/metabolism , Adenoviridae/genetics , Bacteriophage M13/genetics , Bacteriophage lambda/genetics , Bacteriophage phi X 174/genetics , Base Sequence , Binding Sites/genetics , DNA, Viral/genetics , DNA, Viral/metabolism , Deoxyribonucleases, Type II Site-Specific/classification , Deoxyribonucleases, Type II Site-Specific/isolation & purification , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Substrate SpecificityABSTRACT
A new type IIS restriction endonuclease Bfi I hasbeen partially purified from Bacillus firmus S8120. Bfi I recognizes the non-palindromic hexanucleotide sequence 5'-ACTGGG(N)5/4-3' and makes a staggered cut at the fifth base pair downstream of the recognition sequence on the upper strand, producing a single base 3' protruding end.
Subject(s)
Bacillus/enzymology , Deoxyribonucleases, Type II Site-Specific/metabolism , Oligodeoxyribonucleotides/metabolism , Base Sequence , Substrate SpecificityABSTRACT
Bcg I and Bcg I-like restriction endonucleases cleave double stranded DNA specifically on both sides of their asymmetric recognition sequences which are interrupted by several ambiguous base pairs. Their heterosubunit structure, bifunctionality and stimulation by AdoMet make them different from other classified restriction enzymes. Here we report on a new Bcg I-like restriction endonuclease, Bpl I from Bacillus pumilus , which in contrast to all other Bcg I-like enzymes, recognizes a symmetric interrupted sequence, and which, like Bcg I, cleaves double stranded DNA upstream and downstream of its recognition sequence (8/13)GAGN5CTC(13/8). Like Bcg I, Bpl I is a bifunctional enzyme revealing both DNA cleavage and methyltransferase activities. There are two polypeptides in the homogeneous preparation of Bpl I with molecular masses of approximately 74 and 37 kDa. The sizes of the Bpl I subunits are close to those of Bcg I, but the proportion 1:1 in the final preparation is different from that of 2:1 in Bcg I. Low activity observed with Mg2+increases >100-fold in the presence of AdoMet. Even with AdoMet though, specific cleavage is incomplete. S -adenosylhomocysteine (AdoHcy) or sinefungin can replace AdoMet in the cleavage reaction. AdoHcy activated Bpl I yields complete cleavage of DNA.
