A novel suicide substrate for DNA topoisomerases and site-specific recombinases

Burgin, Alex B.; Huizenga, Beth N.; Nash, Howard A.

doi:10.1093/nar/23.15.2973

Cited by 80 publications

(98 citation statements)

References 34 publications

(17 reference statements)

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“…Topo70 was concentrated to 4 mg͞ml in 10 mM Tris⅐HCl, pH 7.5/1 mM EDTA/1 mM DTT. Blunt-ended duplex oligonucleotides were prepared with a 5Ј-bridging phosphorothiolate linkage (12) at the preferred site of topo I cleavage (13). The oligonucleotide sequence was 5Ј-AAAAAGACTTsTGAAAAATTTTT-3Јin the binary topo70-DNA cr ystal form and 5Ј-A A A A AGACT TsG-GAAAAATTTTT-3Ј in the ternary topo70-DNA-Topotecan crystal form, where ''s'' represents the 5Ј bridging phosphorothiolate of the cleaved strand.…”

Section: Methodsmentioning

confidence: 99%

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The mechanism of topoisomerase I poisoning by a camptothecin analog

Staker¹,

Hjerrild²,

Feese³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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We report the x-ray crystal structure of human topoisomerase I covalently joined to double-stranded DNA and bound to the clinically approved anticancer agent Topotecan. Topotecan mimics a DNA base pair and binds at the site of DNA cleavage by intercalating between the upstream (؊1) and downstream (؉1) base pairs. Intercalation displaces the downstream DNA, thus preventing religation of the cleaved strand. By specifically binding to the enzyme-substrate complex, Topotecan acts as an uncompetitive inhibitor. The structure can explain several of the known structure-activity relationships of the camptothecin family of anticancer drugs and suggests that there are at least two classes of mutations that can produce a drug-resistant enzyme. The first class includes changes to residues that contribute to direct interactions with the drug, whereas a second class would alter interactions with the DNA and thereby destabilize the drug-binding site. Eukaryotic DNA topoisomerase I (topo I) is an enzyme that acts to relax supercoils generated during transcription and DNA replication (1). Because of the size of the eukaryotic chromosome, removal of these supercoils can only be accomplished locally by introducing breaks into the DNA helix. Topo I mediates DNA relaxation by creating a transient single-strand break in the DNA duplex. This transient nick allows the broken strand to rotate around its intact complement, effectively removing local supercoils. Strand nicking results from the transesterification of an active-site tyrosine (Tyr-723) at a DNA phosphodiester bond forming a 3Ј-phosphotyrosine covalent enzyme-DNA complex. After DNA relaxation, the covalent intermediate is reversed when the released 5Ј-OH of the broken strand reattacks the phosphotyrosine intermediate in a second transesterification reaction. The rate of religation is normally much faster than the rate of cleavage, and this ensures that the steady-state concentration of the covalent 3Ј-phosphotyrosyl topo I-DNA complex remains low (2).However, a variety of DNA lesions and drugs have been shown to stabilize the covalent 3Ј-phosphotyrosyl intermediate (3). For example, camptothecin (CPT) is a natural product that was originally discovered because of its antitumor activity (4) and was later demonstrated to cause the accumulation of topo I-DNA adducts in vitro and in vivo (5, 6). CPTs bind the covalent 3Ј-phosphotyrosyl intermediate and specifically block DNA religation (7), thus converting topo I into a DNA-damaging agent (8). Topo I is the sole intramolecular target of CPT, and the cytotoxic effects of CPT poisoning are S-phase specific (9). During DNA replication, the replication fork is thought to collide with the ''trapped'' topo I-DNA complexes, resulting in double-strand breaks and ultimately cell death (10).It has been difficult to study the mechanism of CPT activity because the drug acts as an uncompetitive inhibitor and binds only the transient covalent enzyme-substrate complex (7,11). To isolate the covalent topo I-DNA complex, we have used suicide DNA ...

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Section: Methodsmentioning

confidence: 99%

“…To isolate the covalent topo I-DNA complex, we have used suicide DNA substrates containing a 5Ј-bridging phosphorothiolate (12). Topo I-mediated cleavage of these substrates generates a 5Ј-sulfhydryl, instead of a 5Ј-hydroxyl, which is inert in subsequent ligation reactions.…”

mentioning

confidence: 99%

The mechanism of topoisomerase I poisoning by a camptothecin analog

Staker¹,

Hjerrild²,

Feese³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

710

609

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“…The 5Ј bridging phosphorothiolate substrates were synthesized by A. B. Burgin (deCode Biostructures, Bainbridge Island, WA) as described (35,36) and were assembled by ligation of the following annealed oligonucleotides: 5Ј-GATCACTCTATACTA-ATAAAAAATTA*TATAT-3Ј, where * indicates the position of the bridging phosphorothiolate, and 5Ј-ATAATTTTTTAT-TAGTATAGAGTG-3Ј. Annealing of oligomers was carried out by heating equimolar amounts of each oligonucleotide at 95°C for 5 min, followed by slow cooling to room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…However, the end-product assay does not allow a reliable assessment of cleavage activity, because reversibility of the reaction could lead to rapid conversion of the cleaved intermediate back into substrate. Therefore, to directly assay the cleavage ability of the mutant proteins, we used a modified DNA substrate containing 5Ј bridging phosphorothiolates (the 5Ј bridging oxygen is replaced by a sulfur) at the cleavage sites (35,36). With this substrate, cleavage intermediates become trapped in a CPD because the resulting 5Ј sulfhydryl group is a very poor nucleophile and is unable to promote either the forward or reverse ligation step.…”

Section: Rest Mutants In the Hydrophobic Binding Pocket Of The Hairpinmentioning

confidence: 99%

Mixing active-site components: A recipe for the unique enzymatic activity of a telomere resolvase

Bankhead

Chaconas

2004

Proc. Natl. Acad. Sci. U.S.A.

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The ResT protein, a telomere resolvase from Borrelia burgdorferi, processes replication intermediates into linear replicons with hairpin ends by using a catalytic mechanism similar to that for tyrosine recombinases and type IB topoisomerases. We have identified in ResT a hairpin binding region typically found in cut-and-paste transposases. We show that substitution of residues within this region results in a decreased ability of these mutants to catalyze telomere resolution. However, the mutants are capable of resolving heteroduplex DNA substrates designed to allow spontaneous destabilization and prehairpin formation. These findings support the existence of a hairpin binding region in ResT, the only known occurrence outside a transposase. The combination of transposaselike and tyrosine-recombinase-like domains found in ResT indicates the use of a composite active site and helps explain the unique breakage-and-reunion reaction observed with this protein. Comparison of the ResT sequence with other known telomere resolvases suggests that a hairpin binding motif is a common feature in this class of enzyme; the sequence motif also appears in the RAG recombinases. Finally, our data support a mechanism of action whereby ResT induces prehairpin formation before the DNA cleavage step.

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“…The phosphorothiolates were synthesizes as described (186). These suicide oligonucleotides were 5'-end-labeled with [γ- …”

Section: Dna and Suicide Cleavage Assaysmentioning

confidence: 99%

Enzyme Architecture and Flexibility Affect DNA Topoisomerase I Function

Merwe¹

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ii DEDICATION I would like to dedicate this dissertation to my parents, Andries van der Merwe and Marita Esterhuyse for giving me roots so that I can stand strong and to my husband and best friend, Jan Kirsten, for giving me wings so that I can follow my dreams. demonstrate that the conserved core and C-terminal domains dictate the intrinsic enzyme sensitivity to CPT, while it is the functional interactions of the N-terminal and linker domains that regulate enzyme activity in vivo.vi "Since the two chains in our model are intertwined, it is essential for them to untwist if they are to separate. Although it is difficult at the moment to see how these processes occur without everything getting tangled, we do not feel that this objection would be insuperable" (1).In 1953 the scientific duo, Watson and Crick solved the structure of duplex DNA.They found that DNA exists as a double helix and that the bases on each strand were complementary to each other. This helical and complementary character of the DNA double helix represented an elegant solution to the complicated task of housing genetic information. It was further envisioned that to gain access to the information in this molecule, the two strands of the double helix must separate from each other and that the separation will have topological consequences (1). This problem leads to the fundamental need in all cells for a class of enzymes that is able to alter DNA topology, hence DNA topoisomerases. These enzymes are ubiquitous and found in all organisms, including viruses, bacteria, archaebacteria and eukaryotes (2).All DNA topoisomerases alter DNA topology through a cleavage/religation mechanism that employs the chemistry of transesterification. Cleavage is initiated by the Type IA enzymes catalyze DNA strand passage by an "enzyme bridging" mechanism, whereby the enzyme cleaves a single DNA strand and holding onto both DNA ends at the break, bridges the opening through which an intact strand is passed. It does not require an external energy source such as ATP, but Mg(II) is necessary for its relaxation activity.Type IA topoisomerases first bind to a short stretch of single stranded DNA and therefore preferentially relaxes negatively supercoiled or underwound DNA. After binding to the single stranded DNA, the nucleophilic O-4 oxygen of the catalytic Tyr Single molecule experiments have confirmed this model of relaxation by type IA enzymes and further demonstrated that this enzyme is able to increase the linking number of positively supercoiled DNA providing that there is a short stretch of unpaired DNA (11). This demonstrates that the enzyme dictates directionality to the relaxation process.Type IB topoisomerase is distinct from type IA in its mechanism of action as it relaxes DNA by strand rotation. This enzyme binds to duplex DNA and can relax positive or negative supercoils in the absence of energy cofactors or divalent cations. As it relaxes both positive and negative supercoils, the directionality of relaxation is determined by the torsional strain in th...

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A novel suicide substrate for DNA topoisomerases and site-specific recombinases

Cited by 80 publications

References 34 publications

The mechanism of topoisomerase I poisoning by a camptothecin analog

The mechanism of topoisomerase I poisoning by a camptothecin analog

Mixing active-site components: A recipe for the unique enzymatic activity of a telomere resolvase

Enzyme Architecture and Flexibility Affect DNA Topoisomerase I Function

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