Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA

Edwards, K. A.; Halligan, Brian D.; Davis, John L.; Nivera, Nadine L.; Liu, Leroy F.

doi:10.1093/nar/10.8.2565

Cited by 110 publications

(67 citation statements)

References 19 publications

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“…The sequence preferences (if any) for cleavage by DraTopIB are not yet known. It is conceivable that DraTopIB, similar to nuclear TopIB (64,65), is fairly promiscuous in DNA cleavage and might prefer to have a cleavage-ready active site in the free enzyme.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

Patel

Shuman

Mondragón

2006

Journal of Biological Chemistry

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Topoisomerases I and II are involved in virtually all DNA transactions and are the targets of clinically effective anti-cancer and anti-infective drugs (1, 2). Topoisomerases exploit a tyrosine nucleophile to attack the phosphodiester backbone, yielding a covalent enzyme-DNA adduct on one side of the resulting break that permits the passage of strand(s) through the break. Type I enzymes operate by cleaving one DNA strand and passing another strand through the nick; type II enzymes cleave both DNA strands and allow passage of a duplex segment through the double strand break. Closure of the break by reversal of the cleavage step results in a change to the topology of DNA, with no net effect on its chemical structure.Type I topoisomerases are subclassified as type IA or type IB enzymes, depending on whether they form a 5Ј-or 3Ј-phosphotyrosyl adduct, respectively. Type IA enzymes are distributed widely in the bacterial, archaeal, and eukaryal domains of life. Type IB enzymes are found in eukarya, eukaryotic viruses (poxviruses and mimivirus), and many genera of bacteria (1-6). Eukaryotic nuclear type IB enzymes are large monomeric polypeptides, typically Ͼ90 kDa, whereas the viral and bacterial type IB polypeptides are much smaller, typically ϳ33-36 kDa. Despite their differences in size, the poxvirus and nuclear type IB enzymes have a common core tertiary structure and catalytic mechanism (7,8), which is shared with the tyrosine recombinase family (9 -12), thereby suggesting a common ancestry for type IB topoisomerases and tyrosine recombinases (7,8). The active sites of poxvirus and nuclear type IB topoisomerases consist of five conserved functional groups, e.g. Arg-130, Lys-167, Arg-220, His-265, and Tyr-274 in vaccinia virus topoisomerase IB (vaccinia TopIB), which execute the cleavage and religation transesterification steps of the catalytic cycle (13-21).Vaccinia TopIB is composed of two domains separated by a flexible protease-sensitive linker (22, 23). The 234-aa 3 carboxyl-terminal (C) domain contains the active site and catalyzes DNA transesterification and supercoil relaxation but has reduced affinity for DNA compared with the full-length enzyme (24). The 80-aa amino-terminal (N) domain interacts with DNA in the major groove (22,25,26). The atomic structures of the individual domains of vaccinia TopIB have been determined by x-ray crystallography (7,22). Whereas the fold and active site of the predominantly ␣-helical catalytic domain are conserved in nuclear type IB topoisomerases (nuclear TopIB) and the tyrosine recombinases (7), the amino-terminal domain, which consists primarily of ␤-strands (22), has no counterpart in tyrosine recombinases, although it is structurally homologous to part of the much larger amino-terminal domain of nuclear TopIB (27).Bacterial type IB topoisomerases were discovered recently (5) and are similar to poxvirus type IB topoisomerases with respect to their size, primary structures, and domain organization (Fig. 1). Mutational analyses indicate that the transesterification mec...

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Section: Resultsmentioning

confidence: 99%

Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

Patel

Shuman

Mondragón

2006

Journal of Biological Chemistry

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“…3). The rat liver and other eucaryotic type I topoisomerases show a nonrandom pattern of cleavage (3), but cleavage and attachment apparently occur with all four types of nucleotide (3,11). Thus, it will be of interest to determine whether single base changes at the cytidine residues presumed to be the sites of FLP protein attachment to DNA (Fig.…”

Section: Methodsmentioning

confidence: 99%

The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage.

Gronostajski¹,

Sadowski²

1985

Mol. Cell. Biol.

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The FLP recombinase, encoded by the 2,um plasmid of Saccharomyces cerevisiae, promotes efficient recombination in vivo and in vitro between its specific target sites (FLP sites). It was previously determined that FLP interacts with DNA sequences within its target site (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski. Cell 40:795-803, 1985), generates a single-stranded break on both DNA strands within the FLP site, and remains covalently attached to the 3' end of each break. We now show that the FLP protein is bound to the 3' side of each break by an O-phosphotyrosyl residue and that it appears that the same tyrosyl residue(s) is used to attach to either DNA strand within the FLP site.Site-specific recombination plays an important role in a variety of cell-developmental and -regulatory processes. In procaryotes, site-specific inversion events control such diverse functions as flagellar-phase variation (28) and bacteriophage host range specificity (20). In eucaryotes, site-specific recombination events generate deletions of DNA that are required for the production of immunoglobulins (4, 10), the antigen receptors of T-lymphocytes (8, 13), and other products of the immune system (15). The biochemical mechanism of such recombination events is poorly understood, and we have been studying the site-specific recombinase FLP, encoded by the 2,im plasmid of Saccharomyces cerevisiae to gain insight into these processes (1,23,26).The FLP protein promotes efficient recombination between two identical FLP recombination sites (FLP sites) present on the 2,um plasmid in vivo (6) and in vitro (23, 26). In the 2,um plasmid, recombination in vivo between the two sites produces an inversion event so that two forms of the plasmid (A and B) are present within the cell. In vitro, the FLP protein promotes an inversion event when the two FLP sites are in an inverted orientation and a deletion event when the two sites are in a direct orientation (18,23,26). At high concentrations of FLP protein, intermolecular as well as intramolecular recombination occurs (18, R. M. Gronostajski and P. D. Sadowski, J. Biol. Chem., in press). In addition, we have characterized the interaction of the FLP protein with its FLP site DNA target and demonstrated that the protein produces two single-stranded nicks at specific nucleotides in the FLP site to produce an 8-base-pair (bp) staggered break about which recombination presumably occurs (1). Under defined conditions, the protein remains covalently bound to the DNA at the 3' termini of these nicks. Since the 5' termini of such FLP-induced nicks bear free hydroxyl groups, it appears likely that the FLP protein is covalently bound to the DNA via a 3'-phosphoryl linkage. In this report, we present evidence that the FLP recombinase is attached to DNA via a nucleotide 3'-phosphotyrosyl linkage and that the same tyrosyl residue(s) is used for attachment of the protein to either DNA strand. * Corresponding author. MATERIALS AND METHODSPreparation of FLP. FLP protein was purified from Escherichia co...

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“…This difference in template preference may account for the finding that SS DNA is a more potent inhibitor of the bacterial relaxation activity than that of the eucaryotic enzyme (11). Studies of the cleavage of SS DNA using SS and gapped templates also indicate that the procaryotic and eucaryotic enzymes prefer quite different cleavage sites in terms of sequence and potential secondary structure (6,(13)(14)(15)(16)(17). While the bacterial enzymes cleave at SS regions, the eucaryotic enzymes appear to seek regions of duplex structure (18), and hence during strand passage may be less adept at bridging the enzyme-induced break (5,6) than protein u (15,19).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate

Low

Holden

1985

Nucl Acids Res

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The relaxation activity of DNA topoisomerase I from HeLa cell nuclei is strongly inhibited by a variety of purine nucleotides in the presence but not absence of 1 mM potassium phosphate. For ATP, 3-4 nM causes nearly complete inhibition. The 2'-and 3'-AMP isomers are active as well in the presence of 1 mM phosphate, but the 5'-AMP isomer and adenosine are inert. At 3 nM ATP, the titration curve for phosphate is sigmoidal with inhibition beginning abruptly at about 0.5 nM. The negatively-supercoiled DNA isolated from an "inhibited" reaction is relaxed as well as the standard DNA template in the absence of ATP and phosphate suggesting that inhibition does not result from an alteration of the template which protects against its relaxation. Relaxation of positively-supercoiled DNA is also inhibited.Catalysis by E. coli DNA topoisomerase I and HeLa DNA topoisomerase II is not inhibited at concentrations of ATP and phosphate sufficient to cause 80-90% inhibition of HeLa type 1 enzyme.

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Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA

Cited by 110 publications

References 19 publications

Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA

The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage.

Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate

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