Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Pivot Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Target Concepts:
Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
Query: EC:3.1.30.2 (
endonuclease
)
18,621
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
Restriction-modification systems (RMS) are the main gene-engineering tools and a suitable model to study the molecular mechanisms of catalysis and DNA-protein interactions. Research into the catalytic properties of these enzymes, determination of hydrolysis and DNA-methylation sites remain topical. In our previous work we have cloned and sequenced the CfrBI restriction-modification system (strain
Citrobacter freundii)
, which recognizes the nucleotide sequence 5'-CCWWGG-3'. In this article we describe the cloning of the methyltransferase and restriction
endonuclease
genes (gene encoding CfrBI DNA methyltransferase (
cfrBIM)
and gene encoding CfrBI restriction
endonuclease
(
cfrBIR)
) separately to obtain strains overproducing the enzymes of this system. His
6
-CfrBI, which had been purified to homogeneity, was used to establish the DNA-hydrolysis point in its recognition site. CfrBI was shown to cleave DNA after just the first 5'C within the recognition site and then to generate 4-nt 3' cohesive ends (5'-C/CWWGG-3'). To map the site of methylation by M.CfrBI, we exploited the fact that the
Cfr
BI site partially overlaps with the recognition sites of the well-documented enzymes KpnI and ApaI. The M.CfrBI- induced hemimethylation of the internal C residue of the ApaI recognition sequence (GGGC
N4m
CC) was observed to block cleavage by ApaI. In contrast, KpnI was able to digest its M.CfrBI-hemimethylated site (
GGTA
N4m
CC). KpnI was used to restrict a fragment of DNA harbouring the CfrBI and KpnI sites, in which the CfrBI site was methylated
in vitro
by His
6
-M.CfrBI using [
3
H]-SAM. The subsequent separation of hydrolysis products by electrophoresis and the enumeration of incorporated [H3]-methyl groups in each of the fragments made it possible to determine that external cytosine undergoes modification in the recognition site.
...
PMID:An alternative approach to study the enzymatic specificities of the CfrBI restriction-modification system. 3119 72
