Purification of the E.coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethyiguanine and O4-methylthymine in dodecadeoxyribnucleotides

Mark Wilkinson; P. M. Potter; L. Cawkwell; P. Georgiadis; D. Patel; P. F. Swann; G. P. Margison

doi:10.1093/nar/17.21.8475

Purification of the E.coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethyiguanine and O4-methylthymine in dodecadeoxyribnucleotides

Mark Wilkinson, P. M. Potter, L. Cawkwell, P. Georgiadis, D. Patel, P. F. Swann, G. P. Margison

Research output: Contribution to journal › Article › peer-review

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Abstract

The E.coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (̃ 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and (O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG > O6-EtG > O4-MeT, however, the ogt protein was found to repair O6MeG, O6-EtG and (O4-MeT, 1.1, 173 and 84 times, respectively, faster than the ada protein.

Original language	English
Pages (from-to)	8475-8483
Number of pages	9
Journal	Nucleic Acids Research
Volume	17
Issue number	21
DOIs	https://doi.org/10.1093/nar/17.21.8475
Publication status	Published - 11 Nov 1989
Externally published	Yes

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@article{14da78ab128940279401eadbc405ec82,

title = "Purification of the E.coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethyiguanine and O4-methylthymine in dodecadeoxyribnucleotides",

abstract = "The E.coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme {\~(} 0.2\% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and (O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG > O6-EtG > O4-MeT, however, the ogt protein was found to repair O6MeG, O6-EtG and (O4-MeT, 1.1, 173 and 84 times, respectively, faster than the ada protein.",

author = "Mark Wilkinson and Potter, \{P. M.\} and L. Cawkwell and P. Georgiadis and D. Patel and Swann, \{P. F.\} and Margison, \{G. P.\}",

year = "1989",

month = nov,

day = "11",

doi = "10.1093/nar/17.21.8475",

language = "English",

volume = "17",

pages = "8475--8483",

journal = "Nucleic Acids Research",

issn = "0305-1048",

publisher = "Oxford University Press",

number = "21",

}

TY - JOUR

T1 - Purification of the E.coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethyiguanine and O4-methylthymine in dodecadeoxyribnucleotides

AU - Wilkinson, Mark

AU - Potter, P. M.

AU - Cawkwell, L.

AU - Georgiadis, P.

AU - Patel, D.

AU - Swann, P. F.

AU - Margison, G. P.

PY - 1989/11/11

Y1 - 1989/11/11

N2 - The E.coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (̃ 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and (O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG > O6-EtG > O4-MeT, however, the ogt protein was found to repair O6MeG, O6-EtG and (O4-MeT, 1.1, 173 and 84 times, respectively, faster than the ada protein.

AB - The E.coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (̃ 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and (O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG > O6-EtG > O4-MeT, however, the ogt protein was found to repair O6MeG, O6-EtG and (O4-MeT, 1.1, 173 and 84 times, respectively, faster than the ada protein.

U2 - 10.1093/nar/17.21.8475

DO - 10.1093/nar/17.21.8475

M3 - Article

SN - 0305-1048

VL - 17

SP - 8475

EP - 8483

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 21

ER -

Purification of the E.coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethyiguanine and O4-methylthymine in dodecadeoxyribnucleotides

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