Oligonucleotide cleavage by restriction endonucleases MvaI and EcoRII: a comprehensive study on the influence of structural parameters on the enzyme-substrate interaction.
To elucidate the mechanism of action of the restriction endonucleases--isoschizomers EcoRII and MvaI--a study was made of their interaction with a set of synthetic oligonucleotide duplexes containing a single 5'-d(CCA/TGG)-3' EcoRII (MvaI) recognition site. The substrates had varying length and structure of the nucleotide sequences flanking the recognition site. The structure of the flanking sequence is important for the cleavage by EcoRII and MvaI enzymes; there is a structure which was found to speed up the EcoRII and MvaI action. The cleavage of oligonucleotide duplexes by EcoRII enzyme does not go to completion. EcoRII endonuclease cleaved extended substrates less efficiently than short ones. Extension of the flanking sequences, with the same nucleotide surrounding of the recognition site, substantially altered the whole kinetic pattern of MvaI hydrolysis. This was not observed with EcoRII enzyme. The restriction endonuclease MvaI distinguished between dA and dT residues in the recognition site, which was reflected in the higher rate of hydrolysis of the dA-containing strand of the quasi-palindromic DNA duplex.
Kubareva
E A
EA
A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, U.S.S.R.
Gromova
E S
ES
Pein
C D
CD
Krug
A
A
Oretskaya
T S
TS
Cech
D
D
Shabarova
Z A
ZA
D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000737:chemistry*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides / Q000378:metabolism*; D013379:Substrate Specificity
D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D004247:DNA; C029528:DNA modification methylase EcoRII; D015257:DNA-Cytosine Methylases; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific