DNA topoisomerases solve the topological problems associated with DNA replication, transcription, recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. In addition, these enzymes fine-tune the steady-state level of DNA supercoiling both to facilitate protein interactions with the DNA and to prevent excessive supercoiling that is deleterious. In recent years, the crystal structures of a number of topoisomerase fragments, representing nearly all the known classes of enzymes, have been solved. These structures provide remarkable insights into the mechanisms of these enzymes and complement previous conclusions based on biochemical analyses. Surprisingly, despite little or no sequence homology, both type IA and type IIA topoisomerases from prokaryotes and the type IIA enzymes from eukaryotes share structural folds that appear to reflect functional motifs within critical regions of the enzymes. The type IB enzymes are structurally distinct from all other known topoisomerases but are similar to a class of enzymes referred to as tyrosine recombinases. The structural themes common to all topoisomerases include hinged clamps that open and close to bind DNA, the presence of DNA binding cavities for temporary storage of DNA segments, and the coupling of protein conformational changes to DNA rotation or DNA movement. For the type II topoisomerases, the binding and hydrolysis of ATP further modulate conformational changes in the enzymes to effect changes in DNA topology.

Topoisomerases I promote the relaxation of DNA superhelical tension by introducing a transient single-stranded break in duplex DNA and are vital for the processes of replication, transcription, and recombination. The crystal structures at 2.1 and 2.5 angstrom resolution of reconstituted human topoisomerase I comprising the core and carboxyl-terminal domains in covalent and noncovalent complexes with 22-base pair DNA duplexes reveal an enzyme that "clamps" around essentially B-form DNA. The core domain and the first eight residues of the carboxyl-terminal domain of the enzyme, including the active-site nucleophile tyrosine-723, share significant structural similarity with the bacteriophage family of DNA integrases. A binding mode for the anticancer drug camptothecin is proposed on the basis of chemical and biochemical information combined with these three-dimensional structures of topoisomerase I-DNA complexes.

The three-dimensional structure of a 70-kilodalton amino terminally truncated form of human topoisomerase I in complex with a 22-base pair duplex oligonucleotide, determined to a resolution of 2.8 angstroms, reveals all of the structural elements of the enzyme that contact DNA. The linker region that connects the central core of the enzyme to the carboxyl-terminal domain assumes a coiled-coil configuration and protrudes away from the remainder of the enzyme. The positively charged DNA-proximal surface of the linker makes only a few contacts with the DNA downstream of the cleavage site. In combination with the crystal structures of the reconstituted human topoisomerase I before and after DNA cleavage, this information suggests which amino acid residues are involved in catalyzing phosphodiester bond breakage and religation. The structures also lead to the proposal that the topoisomerization step occurs by a mechanism termed "controlled rotation."

The phospholipase D (PLD) superfamily is a diverse group of proteins that includes enzymes involved in phospholipid metabolism, a bacterial toxin, poxvirus envelope proteins, and bacterial nucleases. Based on sequence comparisons, we show here that the tyrosyl-DNA phosphodiesterase (Tdp1) that has been implicated in the repair of topoisomerase I covalent complexes with DNA contains two unusual HKD signature motifs that place the enzyme in a distinct class within the PLD superfamily. Mutagenesis studies with the human enzyme in which the invariant histidines and lysines of the HKD motifs are changed confirm that these highly conserved residues are essential for Tdp1 activity. Furthermore, we show that, like other members of the family for which it has been examined, the reaction involves the formation of an intermediate in which the cleaved substrate is covalently linked to the enzyme. These results reveal that the hydrolytic reaction catalyzed by Tdp1 occurs by the phosphoryl transfer chemistry that is common to all members of the PLD superfamily.T he members of the phospholipase D (PLD) superfamily comprise a highly diverse group of proteins that include plant, mammalian and bacterial PLDs, bacterial phosphatidylserine and cardiolipin synthases, a bacterial toxin, several poxvirus envelope proteins, and some bacterial nucleases (1-3). Sequence alignments reveal that with the exception of two nucleases (1) the proteins arose as a result of a gene duplication event with each half of the protein containing four repeated motifs. Motifs 3 and 4 contain the highly conser ved HxK(x) 4 D(x) 6 GSxN sequence, termed the HKD motif (4), which has been implicated in the catalytic mechanism (see below). The crystal structure of the Salmonella typhimurium Nuc protein (4), one of the bacterial nucleases, shows that the active form of the enzyme is a dimer with the HKD motifs contributed by each subunit organized in roughly the same spatial arrangement as the two HKD motifs found in the crystal structure of the monomeric PLD from Streptomyces sp. strain PMF (5).For those members of the superfamily known to have catalytic activity, the enzymatic reactions all involve phosphoryl transfer from a donor to an acceptor that is either an alcohol or water. In the cases of the phospholipid synthases, a phosphatidyl moiety is transferred either from a cytidine 5Ј-diphosphate-diacylglycerol donor to serine or from phosphatidylglycerol to another phosphatidylglycerol to synthesize phosphatidylserine or cardiolipin, respectively. The PLDs either hydrolyze the phosphodiester bond in the phospholipid to produce phosphatidic acid and a free head group (often choline) or catalyze the exchange of one head group for another (transphosphatidylation). The nucleases appear to simply catalyze the hydrolysis of DNA phosphodiester bonds.The similar chemistry underlying these reactions suggests that the enzymes in the superfamily share a similar catalytic mechanism. Early evidence showing retention of configuration at the substrate phosphorous in the re...

Reconstituted transcription reactions containing the seven general transcription factors, in addition to RNA polymerase II, respond poorly to transcriptional activators. Two factors, Dr2 and ACF, necessary for high levels of transcription in response to an activator have been identified. ACF can enhance basal and activated transcription. Dr2 represses basal transcription, but this can be overcome by transcriptional activators or TFIIA. Dr2 is human DNA topoisomerase I. The DNA relaxation activity of topoisomerase I is dispensable for transcriptional repression. The effect of Dr2 is specific for TATA-box-containing promoters and is mediated by the TATA-binding protein.

Human tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes the phosphodiester bond between a DNA 3 end and a tyrosyl moiety. In eukaryotic cells, this type of linkage is found in stalled topoisomerase I-DNA covalent complexes, and Tdp1 has been implicated in the repair of such complexes in vivo. We confirm here that the Tdp1 catalytic cycle involves a covalent reaction intermediate in which a histidine residue is connected to a DNA 3-phosphate through a phosphoamide linkage. Most surprisingly, this linkage can be hydrolyzed by Tdp1, and unlike a topoisomerase I-DNA complex, which requires modification to be an efficient substrate for Tdp1, the native form of Tdp1 can be removed from the DNA. The spinocerebellar ataxia with axonal neuropathy neurodegenerative disease is caused by the H493R mutant form of Tdp1, which shows reduced enzymatic activity and accumulates the Tdp1-DNA covalent intermediate. The ability of wild type Tdp1 to remove the stalled mutant protein from the DNA likely explains the recessive nature of spinocerebellar ataxia with axonal neuropathy. In addition to its activity on phosphotyrosine and phosphohistidine substrates, Tdp1 also possesses a limited DNA and RNA 3-exonuclease activity in which a single nucleoside is removed from the 3-hydroxyl end of the substrate. Furthermore, Tdp1 also removes a 3 abasic site and an artificial 3-biotin adduct from the DNA. In combination with earlier data showing that Tdp1 can use 3-phosphoglycolate as a substrate, these data suggest that Tdp1 may function to remove a variety of 3 adducts from DNA during DNA repair.Tyrosyl-DNA phosphodiesterase (Tdp1) 2 was discovered as an enzymatic activity from Saccharomyces cerevisiae that specifically hydrolyzes the phosphodiester linkage between the O-4 atom of a tyrosine and a DNA 3Ј-phosphate (1). This type of linkage is typical of the covalent reaction intermediate produced when a type IB topoisomerase cleaves one strand of DNA. Type IB topoisomerases are ubiquitous enzymes that perform essential functions in key cellular processes such as replication, recombination, and transcription (2-6). Following cleavage and DNA relaxation, the topoisomerase normally religates the DNA strand and dissociates from the DNA (4). After cleavage near certain DNA lesions or modified nucleotides, or in the presence of the anticancer drug camptothecin, religation is blocked, and the topoisomerase becomes covalently trapped on the 3Ј end of the cleaved DNA strand (7-10). At the time of the discovery of Tdp1, Yang et al.(1) suggested that the enzyme might be involved in the removal of such covalently stalled topoisomerase I molecules from the DNA. At present, extensive genetic, biochemical, and cell biological data support the view that Tdp1 is involved in the repair of topoisomerase I-DNA covalent lesions in eukaryotic cells (11)(12)(13)(14)(15)(16).Tdp1 is a member of the phospholipase D (PLD) superfamily, which is characterized by two "HKD" motifs that provide the conserved active site residues (17). The diverse members of this family ful...

Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of the tyrosyl-3 0 phosphate linkage found in topoisomerase I-DNA covalent complexes. The inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1), is caused by a H493R mutation in Tdp1. Contrary to earlier proposals that this disease results from a loss-of-function mutation, we show here that this mutation reduces enzyme activity B25-fold and importantly causes the accumulation of the Tdp1-DNA covalent reaction intermediate. Thus, the attempted repair of topoisomerase I-DNA complexes by Tdp1 unexpectedly generates a new protein-DNA complex with an apparent half-life of B13 min that, in addition to the unrepaired topoisomerase I-DNA complex, may interfere with transcription and replication in human cells and contribute to the SCAN1 phenotype. The analysis of Tdp1 mutant cell lines derived from SCAN1 patients reveals that they are hypersensitive to the topoisomerase I-specific anticancer drug camptothecin (CPT), implicating Tdp1 in the repair of CPT-induced topoisomerase I damage in human cells. This finding suggests that inhibitors of Tdp1 could act synergistically with CPT in anticancer therapy.

Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3' phosphate. The enzyme appears to be responsible for repairing the unique protein-DNA linkage that occurs when eukaryotic topoisomerase I becomes stalled on the DNA in the cell. The 1.69 A crystal structure reveals that human Tdp1 is a monomer composed of two similar domains that are related by a pseudo-2-fold axis of symmetry. Each domain contributes conserved histidine, lysine, and asparagine residues to form a single active site. The structure of Tdp1 confirms that the protein has many similarities to the members of the phospholipase D (PLD) superfamily and indicates a similar catalytic mechanism. The structure also suggests how the unusual protein-DNA substrate binds and provides insights about the nature of the substrate in vivo.