Type I topoisomerases

MECHANISM FIGURE 24-21 Bacterial type I topoisomerases alter linking number. A proposed reaction sequence for the bacterial topoi-somerase I is illustrated. The enzyme has closed and open conformations. (a) A DNA molecule binds to the closed conformation and one... 

These synthetic knots are such good substrates for type I topoisomerases that we constructed an RNA trefoil knot to seek RNA topoisomerase activity, which was suspected, but unknown. We designed the knot similarly to the DNA trefoil knot, and made the circle of the same sequence by altering the length of the linker used in the ligation. We discovered that topo HI is capable of catalyzing RNA strand-... 

Catenation by topoisomerases. (a) Two circular DNAs can be catenated by type I topoisomerase only if one of the DNAs is nicked. This is not necessary when using a type II topoisomerase. (b)... 

Type I topoisomerases catalyze the relaxation of supercoiled DNA, a thermodynamically favorable process. Type II topoisomerases utilize free energy from ATP hydrolysis to add negative supercoils to DNA. The two types of enzymes have several common features, including the use of key tyrosine residues to form covalent links to the polynucleotide backbone that is transiently broken. 

The three-dimensional structures of several type I topoisomerases have been determined (Figure 27 21). These structures reveal many features of the reaction mechanism. Human type I topoisomerase comprises four domains, which are arranged around a central cavity having a diameter of 20 A, just the correct size to accommodate a double-stranded DNA molecule. This cavity also includes a tyrosine residue (Tyr 723), which acts as a nucleophile to cleave the DNA backbone in the course of catalysis. 

Supercoiling is catalyzed by type II topoisomerases. These elegant molecular machines couple the binding and hydrolysis of ATP to the directed passage of one DNA double helix through another that has been temporarily cleaved. These enzymes have several mechanistic features in common with the type I topoisomerases. 

The intermediates that form in recombination reactions, with their tyrosine adducts possessing 3 -phosphoryl groups, are reminiscent of the intermediates that form in the reactions catalyzed by topoisomerases. This mechanistic similarity reflects deeper evolutionary relationships. Examination of the three-dimensional structures of recombinases and type I topoisomerases reveals that these proteins are related by divergent evolution despite little amino acid sequence similarity (Figure 27,40). From this perspective, the action of a recombinase can be viewed as an intermolecular topoisomerase reaction. In each case, a tyrosine-DNA adduct is formed. In a topoisomerase reaction, this... 

Champoux, J. (1990) Mechanistic aspects of type-I topoisomerases, in DNA Topology and Its Biological Effects (Wang, J. C. and Cozarelli, N. R., eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 217-242. 

Fig. 2. Reactions of type I topoisomerases. The figure shows the major DNA transformations that can be carried out by type I topoisomerases. Note that catenation or decatenation of circular duplexes will occur only if one DNA molecule bears a single-strand break.

Among the two types of topoisomerases, some enzymes are able to carry out only a subset of the reactions shown in Figs. 2 and 3. For example, the type I topoisomerase from mouse embryo is able to relax positively supercoiled DNA (Champoux and Dulbecco, 1972), whereas... 

The interaction of eucaryotic type I topoisomerases with DNA can also be interrupted by alkali, leading to DNA cleavage (Champoux,... 

Several topoisomerases have been shown to be substrates for protein kinases. Nuclear extracts from a human cell line contain a protein kinase which phosphorylates DNA topoisomerase I from the same cell line (Mills et al., 1982). The type I topoisomerase purified from Novikoff hepatoma cells was found to be a phosphoprotein (Durban et al., 1983). Treatment with alkaline phosphatase dephosphorylates the enzyme and reduces its DNA-relaxing activity. Subsequent treatment with protein kinase restores the activity of the topoisomerase to its original level (Durban et al., 1983). 

This reaction scheme has been extended to other reactions of the type I topoisomerases such as DNA relaxation, knotting, and the annealing of complementary single-strand circles (Brown and Cozzarelli, 1981 Dean et al., 1982 Dean and Cozzarelli, 1985). For this mechanism to operate for all type I reactions, the enzyme must act similarly on nicked and unbroken DNA, and be able to pass either single- or double-stranded DNA through the break (though in the latter case it is not excluded that the strands could be translocated one at a time). 

Type I topoisomerases relax DNA from negative supercoils formed by the action of type 11 topoisomerase by creating transient single-strand breaks in DNA. 

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