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 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 of human topoisomerase I to supercoiled DNA.
Yang
Zheng
Z
Department of Microbiology, School of Medicine, University of Washington, Seattle, WA 98195-7242, USA.
Carey
James F
JF
Champoux
James J
JJ
D004252:DNA Mutational Analysis; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D055503:Protein Multimerization; D017433:Protein Structure, Secondary
D004278:DNA, Superhelical; D004264:DNA Topoisomerases, Type I