Camptothecins Inhibit the Utilization of Hydrogen Peroxide in the Ligation Step of Topoisomerase I Catalysis

Lisby, Michael; Krogh, Berit Olsen; Boege, Fritz; Westergaard, Ole; Knudsen, Birgitta R.

doi:10.1021/bi980757r

Cited by 39 publications

(43 citation statements)

References 37 publications

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“…Plasmid pHT143, for expression of recombinant full-length human topo I (p91) in S. cerevisiae, was described previously (16). Plasmid pHT148, for expression of a fusion of GST to the N terminus of amino acids 207-765 of human topo I (GST-p67), was constructed by first cloning a polymerase chain reaction fragment of pGEX-2TK containing the GST tag and the polylinker into a BamHI/EcoRI fragment of pHT143.…”

Section: Methodsmentioning

confidence: 99%

Residues within the N-terminal Domain of Human Topoisomerase I Play a Direct Role in Relaxation*

Lisby

Olesen

Skouboe

et al. 2001

Journal of Biological Chemistry

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All eukaryotic forms of DNA topoisomerase I contain an extensive and highly charged N-terminal domain. This domain contains several nuclear localization sequences and is essential for in vivo function of the enzyme. However, so far no direct function of the N-terminal domain in the in vitro topoisomerase I reaction has been reported. In this study we have compared the in vitro activities of a truncated form of human topoisomerase I lacking amino acids 1-206 (p67) with the full-length enzyme (p91). Using these enzyme forms, we have identified for the first time a direct role of residues within the N-terminal domain in modulating topoisomerase I catalysis, as revealed by significant differences between p67 and p91 in DNA binding, cleavage, Eukaryotic topoisomerase I (topo I)1 is a monomeric enzyme that plays a major role in important cellular processes by regulating the topology of DNA. The enzyme relaxes negative and positive supercoils arising as a consequence of DNA processes such as DNA transcription, replication, recombination, and chromosome condensation (1). Mechanistically, topo I acts by introducing transient singlestrand breaks into the DNA double helix. The catalytic cycle can be subdivided into several steps including: (i) non-covalent DNA binding, (ii) cleavage, (iii) strand rotation, (iv) religation, and (v) enzyme turnover. The cleavage and religation events constitute two reverse phosphoryl transfer (transesterification) reactions. During the cleavage reaction, an active-site tyrosine residue of the enzyme is used as a nucleophile to break a phosphodiester bond of the DNA backbone, generating a covalent enzyme-(3Ј-phosphotyrosyl)-DNA linkage and a free 5Ј-hydroxyl group (2-4). This 5Ј-hydroxyl group provides the nucleophile for the religation reaction that restores intact DNA.The solved crystal structure of an N-terminal-truncated version of the human topo I together with proteolytic analyses show that the enzyme is organized into four structural domains. These consist of an N-terminal domain (amino acids 1-206), a core domain (amino acids 207-635), a linker domain (amino acids 636 -712), and a C-terminal domain (amino acids 713-765) (2, 3). The C-terminal domain contains the active-site tyrosine (Tyr 723 ), which together with the catalytic residues Arg 488 , Lys 532 , Arg 590 , and His 632 of the core domain constitutes the active site of the enzyme (4 -8). Structural data show that the core and C-terminal domains form a clamp structure that wraps around the DNA and, together with the helix-turnhelix linker domain (1), contacts DNA in a region extending 4 base pairs upstream and 9 base pairs downstream of the cleavage site (3). Based on this structural information of the human topo I-DNA complex, a model for strand rotation (topoisomerization) has been proposed. According to this "controlled rotation" model, rotation of the cleaved strand around the intact strand is partially hindered by contacts between the rotating DNA and part of the core and linker domains (3). The involvement of the linker ...

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Section: Methodsmentioning

confidence: 99%

Residues within the N-terminal Domain of Human Topoisomerase I Play a Direct Role in Relaxation*

Lisby

Olesen

Skouboe

et al. 2001

Journal of Biological Chemistry

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“…Although the conformation of the catalytic pocket of the enzyme may determine the initial interaction with the drug, this interaction could be further reinforced by additional drug-DNA interactions. Consistent with such a mechanism, the presence of DNA downstream to the cleavage site in topo I-DNA complexes has been found not to be a prerequisite for, even though it stimulates, the action of camptothecins (26).…”

Section: Discussionmentioning

confidence: 89%

“…An ϳ50% reduction of activity was observed in the presence of 0.05 mM NSC-314622 (open circles) or 1 mM CPT (filled circles), respectively. In comparison, topo I-mediated ligation is reduced by 50% in the presence of 1 M CPT (26), whereas the inhibition efficiencies of NSC-31422 on topo I and Flp appear comparable (17).…”

Section: Flp-mediated Dna Cleavage and Ligation Are Inhibited Bymentioning

confidence: 89%

“…Rather, the active site of Flp may share sufficient structural similarities to that of eukaryotic topo I to be able to bind the two drugs specifically, although Flp differs from topo I regarding drug affinity. Flp is less sensitive than topo I toward CPT (26), but the effect of NSC-314622 on the two enzymes appears comparable (17). Drug interaction with topo I is very specific as revealed by several single amino acid mutations conferring drug resistance to the enzyme (32)(33)(34)(35)(36) and by topo I from vaccinia virus being drug-resistant because of a single amino acid difference compared with the cellular forms of topo I (37,38).…”

Section: Discussionmentioning

confidence: 99%

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Inhibition of Flp Recombinase by the Topoisomerase I-targeting Drugs, Camptothecin and NSC-314622

Frøhlich

Hansen

Lisby

et al. 2001

Journal of Biological Chemistry

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Recombinases of the -Int family and type IB topoisomerases act by introducing transient single strand breaks in DNA using chemically identical reaction schemes. Recent structural data have supported the relationship between the two enzyme groups by revealing considerable similarities in the architecture of their catalytic pockets. In this study we show that the Int-type recombinase Flp is inhibited by the two structurally unrelated topoisomerase I-directed anti-cancer drugs, camptothecin (CPT) and NSC-314622. The interaction of these drugs with topoisomerase I is very specific with several single amino acid substitutions conferring drug resistance to the enzyme. Thus, the observed interaction of CPT and NSC-314622 with Flp, which is comparable to their interaction with the cleavage complex formed by topoisomerase I, strongly supports a close mechanistic and evolutionary relationship between the two enzymes. The results suggest that Flp and other Int family recombinases may provide model systems for dissecting the molecular mechanisms of topoisomerase I-directed anti-cancer therapeutic agents. Members of the integrase (Int)1 family of site-specific recombinases (-integrase, P1 Cre recombinase, Escherichia coli XerC/XerD recombinase, Saccharomyces cerevisiae Flp, and Zygosaccharomyces rouxii R recombinases among several others) all carry out conservative site-specific recombination using a basic type IB topoisomerase reaction scheme (1, 2). In the first step of recombination, an active site tyrosine nucleophile attacks the target phosphodiester bond in DNA to generate a 3Ј-phosphotyrosyl linkage and a free 5Ј-hydroxyl group. In the second step leading to strand rejoining, the 5Ј-hydroxyl group is the nucleophile, and the 3Ј-phosphotyrosyl bond is its target.The nucleophilic attack is directed across partner substrates so that strand ligation occurs in the recombinant configuration, which is opposed to the typical type IB topoisomerase reaction in which ligation restores the original phosphodiester bond (3). However, the two ligation modes are not mutually exclusive, as evident from the fact that some Int recombinases can relax supercoiled DNA in vitro under certain conditions while some type IB topoisomerases can mediate recombination and resolution of Holliday junctions (4 -8). Recent structural data have further consolidated the relationship between the Int family recombinases and the type IB topoisomerases. Despite the lack of overall sequence homology between the two enzyme groups, the tertiary folds within the catalytic domains are strikingly similar between the Int-type recombinases, including Flp (9) and Cre (10), and eukaryotic topoisomerase I (topo I) (11)(12)(13)(14). Moreover, the critical catalytic moieties include two nearly identical tetrads, RHRH/W in the recombinases and RKRH in the topoisomerases, together with the invariant tyrosine nucleophile (1,11).In this study we have further probed the functional relationship between the Int family recombinase and type IB topoisomerase active sites by inves...

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“…RPA was bacterially expressed and purified as outlined before (18,34,45). Human topo I expressed in yeast and purified as described by Lisby et al (24) was a generous gift of M. Lisby, University of Århus, Denmark. Monoclonal antibodies SJK237-71, SJK287-38 (46), F5 (36), and PAb101 (15, 16) specific for PyV and SV40 Tag were purified by affinity chromatography (17).…”

Section: Methodsmentioning

confidence: 99%

Different Regions of Primase Subunit p48 Control Mouse Polyomavirus and Simian Virus 40 DNA Replication In Vitro

Kautz

Schneider

Weißhart

et al. 2001

J Virol

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DNA polymerase ␣-primase (pol-prim), a complex consisting of four subunits, is the major species-specific factor for mouse polyomavirus (PyV) and simian virus 40 (SV40) DNA replication. Although p48 is the most conserved subunit of pol-prim, it is required for in vitro PyV DNA replication but can inhibit cell-free SV40 DNA replication. Production of chimeric human-mouse p48 revealed that different regions of p48 are involved in supporting PyV DNA replication and inhibiting SV40 DNA replication. The N and C-terminal parts of p48 do not have species-specific functions in cell-free PyV DNA replication, but the central part (amino acids [aa] 129 to 320) controls PyV DNA replication in vitro. However, PyV T antigen physically binds to mouse, human, and chimeric pol-prim complexes independently, whether they support PyV DNA replication or not. In contrast to the PyV system, the inhibitory effects of mouse p48 on SV40 DNA replication are mediated by N-and C-terminal regions of p48. Thus, a chimeric p48 containing human aa 1 to 128, mouse aa 129 to 320, and human aa 321 to 418 is active in both PyV and SV40 DNA replication in vitro.Papovaviruses are small DNA tumor viruses (47). For their DNA replication these viruses contribute a viral origin of replication (ori) and the large tumor antigens (Tag) of mouse polyomavirus (PyV) and simian virus 40 (SV40) or the E1 and E2 proteins of papillomaviruses; all other replication factors are supplied by the host (6,42,47,49). Therefore, papovaviral DNA replication in vivo and in vitro has served as a model system to study virus-host interactions and the mechanisms of DNA replication in mammalian cells (37,47,49).These studies allowed the development of a model for eukaryotic DNA replication which relies on unwinding of doublestranded (ds) DNA and stepwise assembly of multiprotein complexes mediated by the viral initiator of DNA synthesis, large Tag. After Tag has recognized and formed a double hexamer at the replication origin, specific distortions of the dsDNA occur (10, 25). The following steps of DNA replication require the specific recruitment of host replication proteins, such as DNA polymerase ␣-primase (pol-prim), eukaryotic single-stranded (ss) DNA-binding protein, replication protein A (RPA), and topoisomerase I (topo I), to the origin of replication (7,11,12,14,26,27,39,40,41). The interaction of Tag with pol-prim stimulates ori binding by Tag (29). Melting and unwinding of ori dsDNA by Tag's helicase activity requires stabilization of the ssDNA by RPA. The melting of ori DNA by Tag also needs the relaxation of supercoiled DNA by topo I. The first RNA primer is then synthesized by the primase activity of pol-prim. The preinitiation and initiation steps of viral DNA synthesis at the origin of DNA replication require, in addition to the DNA-binding and enzymatic activities of the replication factors, specific physical contacts between these protein complexes (11,48,50,51). The newly synthesized RNA is elongated by the DNA polymerase activity of pol-prim. After a replica...

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Camptothecins Inhibit the Utilization of Hydrogen Peroxide in the Ligation Step of Topoisomerase I Catalysis

Cited by 39 publications

References 37 publications

Residues within the N-terminal Domain of Human Topoisomerase I Play a Direct Role in Relaxation*

Residues within the N-terminal Domain of Human Topoisomerase I Play a Direct Role in Relaxation*

Inhibition of Flp Recombinase by the Topoisomerase I-targeting Drugs, Camptothecin and NSC-314622

Different Regions of Primase Subunit p48 Control Mouse Polyomavirus and Simian Virus 40 DNA Replication In Vitro

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