NORMAL GENOMIC

• Redinbo MR, Stewart L, Champoux JJ, Hol WG
• Structural flexibility in human topoisomerase I revealed in multiplenon-isomorphous crystal structures.
• J Mol Biol. 1999; 292: 685-96
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• Human topoisomerase I plays a critical role in chromosomal stability byrelaxing the DNA superhelical tension that arises from a variety ofnuclear processes, including replication, transcription, and chromatinremodeling. Human topoisomerase I is a approximately 91 kDa enzymecomposed of four major domains: a 24 kDa N-terminal domain, a56 kDa coredomain, a7 kDa linker domain, and a6 kDa C-terminal domain containing theactive-site Tyr723 residue. A monoclinic crystal structure of a 70 kDaN-terminally truncated form of human topoisomerase I in non-covalentcomplex with a 22 bp DNA duplex exhibited remarkable crystal-to-crystalnon-isomorphism; shifts in cell constants of up to 9 A in the b -axislength and up to 8.5 degrees in the beta-angle were observed. Eightcrystal structures of human topoisomerase I - DNA complexes from thiscrystal form were determined to between 2.8 and 3.25 A resolution. Thesestructures revealed both dramatic shifts in crystal packing andfunctionally suggestive regions of conformational flexibility in thestructure of the enzyme. Crystal packing shifts of up to 20.5 A combinedwith rotations of up to 11.5 degrees were observed, helping to explain thevariability in cell constants. When the core subdomain III regions of theeight structures are superimposed, the "cap" (core subdomains I and II) ofthe molecule is observed to rotate by up to 4.6 degrees and to shift by upto 3.6 A. The linker domain shows the greatest degree of conformationalflexibility, rotating and shifting by up to 2.5 degrees and 4.6 A,respectively, in five of eight structures, and becoming disorderedaltogether in the remaining three. These observed regions ofconformational flexibility in the cap and the linker domain are consistentwith the structural flexibility invoked in the "controled rotation"mechanism proposed for the relaxation of DNA superhelical tension by humantopoisomerase I.

• Fan Y, Weinstein JN, Kohn KW, Shi LM, Pommier Y
• Molecular modeling studies of the DNA-topoisomerase I ternary cleavablecomplex with camptothecin.
• J Med Chem. 1998; 41: 2216-26
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• The present studies provide a three-dimensional model for the postulatedternary cleavable complex of topoisomerase I (top1), DNA, and camptothecin(CPT). Molecular simulations were done using the AMBER force field. Theresults suggest that a ternary cleavable complex might be stabilized byseveral hydrogen bonds in the binding site. In this proposed"drug-stacking" model, CPT is pseudointercalated in the top1-linked DNAcleavage site and interacts with the protein near its catalytic tyrosinethrough hydrogen bonding and stacking. The structural model is consistentwith the following experimental observations: (i) the N3 position of the5' terminal purine of the cleaved DNA strand is readily alkylated by7-chloromethyl 10,11-methylenedioxy CPT; (ii) CPT generally toleratessubstituents at positions 7, 9, and 10 but is inactivated by additions atposition 12; (iii) 10,11-methylenedioxy (MDO) CPT is much more potent than10,11-dimethoxy (DMO) CPT; (iv) the lactone portion of CPT is essentialfor top1 inhibitory activity; (v) 20S derivatives of CPT are much morepotent than the 20R analogues; (vi) a catalytic tyrosine hydroxyl in top1covalently links to the 3' terminal base, T, of the cleaved DNA strand;and (vii) top1 mutation Asn722Ser leads to CPT resistance. A total of 18camptothecin derivatives with different DNA cleavage potencies were dockedinto the hypothetical cleavable complex binding site to test and refinethe model. These studies provide insight into a possible mechanism of top1inhibition by CPT derivatives and suggest rational approaches for thedesign of new CPT derivatives.

• Katayanagi K et al.
• Three-dimensional structure of ribonuclease H from E. coli.
• Nature. 1990; 347: 306-9
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• The three-dimensional structure of RNase H from Escherichia coli wasdetermined at 1.8 A resolution by X-ray crystallography. The enzyme wasfound to belong to the alpha + beta class of structures, consisting of twodistinct domains. The structure implies a possible region interacting witha DNA-RNA hybrid. The Mg2(+)-binding site essential for activity islocated near a cluster of four acidic amino acids--one glutamic and threeaspartic acid residues. These residues are completely conserved in thehomology alignment of sequences of RNase H and reverse transcriptases fromretroviruses and retrovirus-like entities. The structural motif of betastrands around the Mg2(+)-binding site has similarities to that in DNaseI.

• Been MD, Burgess RR, Champoux JJ
• DNA strand breakage by wheat germ type 1 topoisomerase.
• Biochim Biophys Acta. 1984; 782: 304-12
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• Properties of strand breakage in duplex and single-stranded DNA by thewheat germ type 1 DNA topoisomerase were investigated. Strand breakage induplex DNA is dependent upon the use of denaturing conditions toinactivate the enzyme and terminate the reaction, whereas breakage ofsingle-stranded DNA occurs under the normal reaction conditions and is notdependent upon denaturation. Breakage generates a free 5' hydroxyl groupand enzyme bound to the 3' side of the break, presumably via the 3'phosphate group. The location of sites of breakage with both duplex andsingle-stranded DNA is not random. In all these respects the wheat germenzyme closely resembles the rat liver type 1 topoisomerase. A comparisonof the locations of the sites of breakage in duplex DNA generated by thetopoisomerases from wheat germ and rat liver indicates a number of commonsites, although the patterns of breakage are not identical.