![]() Certain conserved amino acid sequence motifs within this region of DNA topoisomerase II have been predicted to constitute a nucleotide-binding motif ( 36), whereas in bacterial DNA gyrase, residues within these sequence motifs can be mutated to confer resistance to quinolones that stabilize the covalent DNA gyrase/DNA complex ( 37). In type II enzymes, the B′ region is known to be important for DNA binding and cleavage ( 4, 34, 35). The proximity of domain I to the active-site tyrosine and its topological similarity to known nucleotide-binding domains have led to the proposal that it assists in binding the 3′-OH end of the DNA after cleavage by the tyrosine ( 21). In DNA topoisomerase I, this domain contacts domains III and IV directly and lies just to one side of the active-site tyrosine. 4 a) this fold resembles the “Rossmann fold” found in a number of proteins known to bind nucleotides, phosphorylated amino acids, or other small organic molecules ( 22). In both proteins, the βαβαβαβα fold has the same order and relative positioning of secondary structural elements (Fig. The central lobe of the B′ domain of DNA topoisomerase II and domain I of DNA topoisomerase I are both α-β structures, with a four-stranded, parallel β-sheet sandwiched between two pairs of α-helices. Domain I of DNA Topoisomerase I and the B′ Region of DNA Topoisomerase II Are Based on a Similar Fold. Thus, the common use of twofold or pseudo twofold-related CAP domains and certain conserved residues for DNA binding and cleavage by type IA and type II enzymes may reflect a fundamentally similar organization of the catalytic complex. coli DNA topoisomerase I or of Arg-781 of yeast DNA topoisomerase II to alanine results in reduced DNA relaxation and cleavage activity (ref. In support of this idea, mutation of Arg-321 of the E. coli DNA topoisomerase I, Arg-781 in yeast DNA topoisomerase II, and Arg-121 in GyrA), suggesting that this residue may have a role in DNA binding or cleavage. Type IA and type II enzymes also have an absolutely conserved arginine next to the active-site tyrosine (Arg-321 in E. Both type IA and type II enzymes generate 5′ phosphotyrosine linkages to DNA, and it is likely that the active-site tyrosines attack the DNA backbone from a similar local geometry. The similar orientation of the type IA and type II CAP-like domains also may explain their common DNA cleavage polarity. 2), as well as on other CAP-like members such as histone H5 (not shown) ( 30). 2), but they also superpose relatively well on domains III and IV of DNA topoisomerase I (Fig. As expected, the type II CAP-like folds superpose on each other quite well (Table 1, Fig. Table 1 shows the results of superpositions of segments of the CAP-like domains from DNA topoisomerase I, DNA topoisomerase II, and GyrA. Moreover, in domain III, although the adjacent β-strands are too short to meet the Kabsch and Sander secondary structure criteria ( 29), the central HTH motif is nonetheless very clear. Domain IV of DNA topoisomerase I and the CAP-like domains of DNA topoisomerase II and GyrA have the same αβααββ order as CAP, but domain III of DNA topoisomerase I is βααββα. This loop is in an analogous position to the “wing” of the “winged-helix” motif, a substructure that is found in certain CAP-like domains such as HNF-3 ( 28) and that contacts the DNA phosphodiester backbone. The active-site tyrosines of these enzymes are always on an extended loop that connects the adjacent β-strands. coli DNA topoisomerase I and the A′ fragments of DNA topoisomerase II and GyrA all contain α-β regions with CAP-like folds ( 4, 24). It has been observed previously that domains III and IV of E. Spatial Relationship of CAP-Like Domains that Bear the Active-Site Tyrosines. The yeast DNA topoisomerase II structure may represent a state similar to the one in which a DNA duplex has been cleaved and the two halves separated, whereas the GyrA structure appears to be a state that can bind uncleaved duplex DNA ( 4, 5). The conformations of the A′ regions in the yeast DNA topoisomerase II and GyrA structures are different, such that the spatial relationship of the active-site tyrosines is changed. The yeast DNA topoisomerase II structure also contains nearly one-half of the B region, residues 419–628, termed B′. These binding and cleavage cores are termed the A′ fragments. 1) comprise most of the A regions of the enzymes, including residues 678–1177 of the yeast enzyme and residues 30–522 of GyrA. In eukaryotic type II topoisomerases, the A and B subunits are part of a single polypeptide chain, and the enzymes are (B-A) 2 dimers, with the B region N-terminal to the A region. The B subunit contains the ATPase activity, whereas the A subunit contains the DNA-cleaving, active-site tyrosine. In prokaryotes, type II enzymes are A 2B 2 tetramers.
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