Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alky It ransferase cDNA

• Published in: Nucleic acids research Vol. 20; no. 8; pp. 1891 - 1895
• Main Authors: Rafferty, Joseph A., Elder, Rhoderick H., Watson, Amanda J., Cawkwell, Lynn, Potter, Philip M., Margison, Geoffrey P.
• Format: Journal Article
• Language: English
• Published: Oxford University Press 25.04.1992
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/20.8.1891

The cDNA encoding Chinese hamster O6-alkylguanine-DNA-alkyltransferase (ATase) has been isolated from a library prepared from RNA isolated from V79 lung flbroblasts which had an upregulated level of this repair activity following stepwlse 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-PA-GE 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-termlnal 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 eprtope 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.