Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA.
The cDNA encoding Chinese hamster O6-alkylguanine-DNA-alkyltransferase (ATase) has been isolated from a library prepared from RNA isolated from V79 lung fibroblasts which had an upregulated level of this repair activity following stepwise selection with a chloroethylating agent (1, 2). Expression of the cDNA in E. coli produced functionally active ATase at levels of 2.5% of total cellular protein as determined by in vitro assay. The recombinant hamster protein has a molecular weight of 28 kDa as estimated by SDS-PAGE and fluorography and this was identical to that in the upregulated cells. The characteristic PCHRV pentapeptide of the alkyl acceptor site has been identified and there is a 68 amino acid residue region which is 90% conserved across all the mammalian proteins so far analysed: in contrast, the N- and C-terminal domains diverge by as much as 50% between species. Polyclonal antibodies to the human and rat ATases hybridised to the hamster protein on western analysis suggesting at least one common epitope shared across species. However, in antibody inhibition experiments neither of the antisera cross reacted with the hamster ATase in a way which interfered with functional activity whereas the anti-human antibodies inhibited the human ATase and the anti-rat antibodies inhibited the rat and mouse ATases. There may therefore be significant tertiary structural differences between the hamster protein and the other mammalian ATases.
Rafferty
J A
JA
CRC Department of Chemical Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.
Elder
R H
RH
Watson
A J
AJ
Cawkwell
L
L
Potter
P M
PM
Margison
G P
GP
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004260:DNA Repair / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D006147:Guanine / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014166:Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D006147:Guanine; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C050956:DNA alkyltransferase