Characterization of Ustilago maydis topoisomerase I: Purification, cloning, biochemical and kinetic analysis
Type I topoisomerases act by transiently breaking one DNA strand, thus allowing the removal of one supercoil. This study is focused on the purification and characterization of the activity of the type I topoisomerase from Ustilago maydis (U. maydis) and its possible role as an enzyme involved in DNA recombination. The U. maydis topoisomerase I represents an excellent model enzyme in the general characterization of higher eukaryotic type I topoisomerases because of the sequence and structural homology between the U. maydis and human topoisomerase I. As highly purified preparation of the native U. maydis topoisomerase I was obtained and enabled the analysis of the activity of this enzyme in the presence of cofactors, characterization of its activity in association with other DNA binding proteins, and most importantly the quantitative and qualitative characterization of the binding of U. maydis topoisomerase I to DNA substrates containing various secondary structures. The U. maydis TOP1 gene encoding DNA topoisomerase I was cloned by amplifying a gene fragment using the polymerase chain reaction and subsequent screening of genomic DNA library using this fragment. The predicted peptide sequence exhibited 30-40% identity to other eukaryotic TOP1 genes, yet differed in two major features. First, an unusually long acidic region was identified near the amino terminus (28/29 residues are acidic), which resembles other nucleolar peptide motifs. Second an atypical carboxy-terminal 'tail', absent in other TOP1 genes, followed the tyrosine residues of the active site. A top1 gene disruption mutant was constructed by replacing the genomic TOP1 gene, with top1::HygR null allele. This mutant lost the abundant topoisomerase I activity evident on wild-type U. maydis, and displayed a subtle beige coloration phenotype evident during cell senescence. U. maydis TOP1 gene was also expressed in Escherchia coli and yeast cells and topoisomerase I activity was analyzed. The activity of topoisomerase I from the lower eukaryote U. maydis was examined in the presence of chromatin associated proteins in a homogeneous system. H1 histone purified from U. maydis and prebound to DNA stimulated topoisomerase I activity in the presence of MgCl$\sb2$. Covalent complex formation, an intermediate step in the relaxation cycle, was not influenced by the presence of H1 histone. The nonhistone high mobility group protein (U. maydis HMP), an analog of HMG17, also stimulated topoisomerase I mediated DNA relaxation. Topoisomerase I was found to relax DNA by either processive mode (MgCl$\sb2$, H1 histone) or distributive mode (HMP). The binding affinity of purified native U. maydis topoisomerase I enzyme for radiolabeled DNA substrates with various secondary structures was determined by gel shift and equilibrium binding analysis. Topoisomerase I exhibited cooperativity in binding to DNA regardless of the substrate structure. Further analysis demonstrated that cruciform DNA has two populations of binding sites for topoisomerase I while the other substrate (single stranded DNA, DNA molecules containing 6 or 1 mismatched base pairs, DNA hairpins, and fully homologous duplex DNA) have a single population of binding sites. The affinity of topoisomerase I for cruciform was found to be an order of magnitude higher than for any of the other substrates. (Abstract shortened by UMI.)
Thiyagarajan, Manimekalai Mohanasundari, "Characterization of Ustilago maydis topoisomerase I: Purification, cloning, biochemical and kinetic analysis" (1997). ETD Collection for Thomas Jefferson University. AAI9730325.