tailieunhanh - Báo cáo khoa học: Mutational analysis of the preferential binding of human topoisomerase I to supercoiled DNA

Human topoisomerase I binds DNA in a topology-dependent fashion with a strong preference for supercoiled DNAs of either sign over relaxed circu-lar DNA. One hypothesis to account for this preference is that a second DNA-binding site exists on the enzyme that mediates an association with the nodes present in supercoiled DNA. | ễFEBS Journal Mutational analysis of the preferential binding of human topoisomerase I to supercoiled DNA Zheng Yang James F. Carey and James J. Champoux Department of Microbiology Schoolof Medicine University of Washington Seattle WA USA Keywords competition binding assay DNA topology node binding supercoiled DNA topoisomerase I Correspondence J. J. Champoux Department of Microbiology University of Washington Box 357242 Seattle WA 98195-7242 UsA Fax 1 206 543 8297 Tel 1 206 543 8574 E-mail champoux@ Received 7 July 2009 revised 9 August 2009 accepted 11 August 2009 doi Human topoisomerase I binds DNA in a topology-dependent fashion with a strong preference for supercoiled DNAs of either sign over relaxed circular DNA. One hypothesis to account for this preference is that a second DNA-binding site exists on the enzyme that mediates an association with the nodes present in supercoiled DNA. The failure of the enzyme to dimerize even in the presence of DNA appears to rule out the hypothesis that two binding sites are generated by dimerization of the protein. A series of mutant protein constructs was generated to test the hypotheses that the homeodomain-like core subdomain II residues 233-319 provides a second DNA-binding site or that the linker or basic residues in core subdomain III are involved in the preferential binding to supercoiled DNAs. When putative DNA contact points within core subdomain II were altered or the domain was removed altogether there was no effect on the ability of the enzyme to recognize supercoiled DNA as measured by both a gel shift assay and a competition binding assay. However the preference for supercoils was noticeably reduced for a form of the enzyme lacking the coiled-coil linker region or when pairs of lysines were changed to glutamic acids in core subdomain III. The results obtained implicate the linker and solvent-exposed basic residues in core subdomain III in the preferential binding