DNA topoisomerase I from Mycobacterium smegmatis: an enzyme with distinct features

A type I topoisomerase has been purified to homogeneity from Mycobacterium smegmatis. It is the largest single subunit enzyme of this class having molecular mass of 110 kDa. The enzyme is Mg2+ dependent and can relax negatively supercoiled DNA, catenate, and knot single-stranded DNA, thus having typical properties of type I topoisomerases. Furthermore, the enzyme makes single-stranded nicks and the 5'-phosphoryl end of the nicked DNA gets covalently linked with a tyrosine residue of the enzyme. However, M. smegmatis enzyme shows some distinctive features from the prototype Escherichia coli topoisomerase I. The enzyme is relatively stable at higher temperatures and not inhibited by spermidine. It apparently does not contain any bound Zn2+ and on modification of cysteine residues retains the activity, suggesting the absence of the zinc-finger motif in DNA binding. Partially purified Mycobacterium tuberculosis topoisomerase I exhibits very similar properties with respect to size, stability, and reaction characteristics. Sequence comparison of topoisomerase I from E. coli and M. tuberculosis shows the absence of zinc-finger motifs in mycobacterial enzyme. Using a two-substrate assay system, we demonstrate that the enzyme acts processively at low ionic strength and switches over to distributive mode at high Mg2+ concentration. Significantly, the enzyme activity is stimulated by single strand DNA-binding protein. There is a potential to exploit the characteristics of the enzyme to develop it as a molecular target against mycobacterial infections

Cyclin D3-associated Kinase Activity Is Regulated by p27(kip1) in BALB/c 3T3 Cells

We report that cyclin D3/cdk4 kinase activity is regulated by p27(kip1) in BALB/c 3T3 cells. The association of p27(kip1) was found to result in inhibition of cyclin D3 activity as measured by immune complex kinase assays utilizing cyclin D3-specific antibodies. The ternary p27(kip1)/cyclin D3/cdk4 complexes do exhibit kinase activity when measured in immune complex kinase assays utilizing p27(kip1)-specific antibodies. The association of p27(kip1) with cyclin D3 was highest in quiescent cells and declined upon mitogenic stimulation, concomitantly with declines in the total level of p27(kip1) protein. The decline in this association could be elicited by PDGF treatment alone; this was not sufficient, however, for activation of cyclin D3 activity, which also required the presence of factors in platelet-poor plasma in the culturing medium. Unlike cyclin D3 activity, which was detected only in growing cells, p27(kip1) kinase activity was present throughout the cell cycle. Since we found that the p27(kip1) activity was dependent on cyclin D3 and cdk4, we compared the substrate specificity of the active ternary complex containing p27(kip1) and the active cyclin D3 lacking p27(kip1) by tryptic phosphopeptide mapping of GST-Rb phosphorylated in vitro and also by comparing the relative phosphorylation activity toward a panel of peptide substrates. We found that ternary p27(kip1)/cyclin D3/cdk4 complexes exhibited a different specificity than the active binary cyclin D3/cdk4 complexes, suggesting that p27(kip1) has the capacity to both inhibit cyclin D/cdk4 activity as well as to modulate cyclin D3/cdk4 activity by altering its substrate preference

p27(Kip1) and p21(Cip1) Are Not Required for the Formation of Active D Cyclin-cdk4 Complexes

Our studies address questions pertaining to the regulation of D cyclin-cdk4 activity, and the following results were obtained. Conditions that increased the abundance of the D cyclins also increased the abundance of enzymatically active D cyclin-cdk4 complexes in mouse embryo fibroblasts (MEFs) lacking both p27(Kip1) and p21(Cip1) (p27/p21(−/−)). Such conditions included ectopic expression of cyclin D1 and inhibition of D cyclin degradation by the proteasome inhibitor MG132. However, as determined by treatment of wild-type MEFs with MG132, maximal accumulation of D cyclin-cdk4 complexes required p27(Kip1) and p21(Cip1) and coincided with the formation of inactive D cyclin-cdk4-p27(Kip1) or -p21(Cip1) complexes. p27(Kip1) or p21(Cip1) also increased the abundance of D cyclin-cdk4 complexes and reduced amounts of cdk4 activity when ectopically expressed in p27/p21(−/−) MEFs. Lastly, increases in the stability of the D cyclins accounted for their greater abundance in wild-type MEFs than in p27/p21(−/−) MEFs. We conclude that (i) D cyclin-cdk4 complexes are formed and become active in the absence of p27(Kip1) and p21(Cip1) and (ii) p27(Kip1) and p21(Cip1) maximize the accumulation but inhibit the activity of D cyclin-cdk4 complexes. We suggest that D cyclin-cdk4 complexes are more stable when bound to p27(Kip1) or p21(Cip1) and that formation of ternary complexes also stabilizes the D cyclins

Differential positioning of functional cysteine residue (s) determines catalytic characteristics of adenosine kinase from divergent sources

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