NORMAL GENOMIC

• Augustin MA, Huber R, Kaiser JT
• Crystal structure of a DNA-dependent RNA polymerase (DNA primase).
• Nat Struct Biol. 2001; 8: 57-61
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• Primases are essential components of the DNA replication apparatus in every organism. They catalyze the synthesis of oligoribonucleotides on single-stranded DNA, which subsequently serve as primers for the replicative DNA polymerases. In contrast to bacterial primases, the archaeal enzymes are closely related to their eukaryotic counterparts. We have solved the crystal structure of the catalytic primase subunit from the hyperthermophilic archaeon Pyrococcus furiosus at 2.3 A resolution by multiwavelength anomalous dispersion methods. The structure shows a two-domain arrangement with a novel zinc knuckle motif located in the primase (prim) domain. In this first structure of a complete protein of the archaeal/eukaryotic primase family, the arrangement of the catalytically active residues resembles the active sites of various DNA polymerases that are unrelated in fold.

• Koonin EV, Wolf YI, Kondrashov AS, Aravind L
• Bacterial homologs of the small subunit of eukaryotic DNA primase.
• J Mol Microbiol Biotechnol. 2000; 2: 509-12

• Grishin NV
• C-terminal domains of Escherichia coli topoisomerase I belong to the zinc-ribbon superfamily.
• J Mol Biol. 2000; 299: 1165-77
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• Detection of remote evolutionary connections is increasingly difficult with sequence and structural divergence. A combination of sequence and structural analysis, in which statistically supported sequence similarity had a crucial impact, revealed that Escherichia coli topoisomerase I C-terminal fragment is evolutionarily related to the three tetracysteine zinc-binding domains of the enzyme. Spatial structure analysis of this C-terminal fragment indicates that it consists of two structurally similar domains and suggests homology between them. Sequence similarity between the zinc-binding domains of type Ia topoisomerases and transcription regulators of known spatial structure helps to conclude that E. coli topo I contains five copies of a zinc ribbon domain at the C terminus. Two of these domains, corresponding to the C-terminal fragment, lost their cysteine residues and are probably not able to bind zinc. Present analyses lead to the classification of the C-terminal fragment of E. coli topoisomerase I as a member of zinc ribbon superfamily, despite the absence of zinc-binding sites.

• Li Z, Mondragon A, Hiasa H, Marians KJ, DiGate RJ
• Identification of a unique domain essential for Escherichia coli DNA topoisomerase III-catalysed decatenation of replication intermediates.
• Mol Microbiol. 2000; 35: 888-95
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• A 17-amino-acid residue domain has been identified in Escherichia coli DNA topoisomerase III (Topo III) that is essential for Topo III-mediated resolution of DNA replication intermediates in vitro. Deletion of this domain reduced Topo III-catalysed resolution of DNA replication intermediates and decatenation of multiply linked plasmid DNA dimers by four orders of magnitude, whereas reducing Topo III-catalysed relaxation of negatively supercoiled DNA substrates only 20-fold. The presence of this domain has been detected in multiple plasmid-encoded topoisomerases, raising the possibility that these enzymes may also be decatenases.

• Mondragon A, DiGate R
• The structure of Escherichia coli DNA topoisomerase III.
• Structure Fold Des. 1999; 7: 1373-83
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• BACKGROUND: DNA topoisomerases are enzymes that change the topology of DNA. Type IA topoisomerases transiently cleave one DNA strand in order to pass another strand or strands through the break. In this manner, they can relax negatively supercoiled DNA and catenate and decatenate DNA molecules. Structural information on Escherichia coli DNA topoisomerase III is important for understanding the mechanism of this type of enzyme and for studying the mechanistic differences among different members of the same subfamily. RESULTS: The structure of the intact and fully active E. coli DNA topoisomerase III has been solved to 3.0 A resolution. The structure shows the characteristic fold of the type IA topoisomerases that is formed by four domains, creating a toroidal protein. There is remarkable structural similarity to the 67 kDa N-terminal fragment of E. coli DNA topoisomerase I, although the relative arrangement of the four domains is significantly different. A major difference is the presence of a 17 amino acid insertion in topoisomerase III that protrudes from the side of the central hole and could be involved in the catenation and decatenation reactions. The active site is formed by highly conserved amino acids, but the structural information and existing biochemical and mutagenesis data are still insufficient to assign specific roles to most of them. The presence of a groove in one side of the protein is suggestive of a single-stranded DNA (ssDNA)-binding region. CONCLUSIONS: The structure of E. coli DNA topoisomerase III resembles the structure of E. coli DNA topoisomerase I except for the presence of a positively charged loop that may be involved in catenation and decatenation. A groove on the side of the protein leads to the active site and is likely to be involved in DNA binding. The structure helps to establish the overall mechanism for the type IA subfamily of topoisomerases with greater confidence and expands the structural basis for understanding these proteins.

• Nichols MD, DeAngelis K, Keck JL, Berger JM
• Structure and function of an archaeal topoisomerase VI subunit with homology to the meiotic recombination factor Spo11.
• EMBO J. 1999; 18: 6177-88
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• In all organisms, type II DNA topoisomerases are essential for untangling chromosomal DNA. We have determined the structure of the DNA-binding core of the Methanococcus jannaschii DNA topoisomerase VI A subunit at 2.0 A resolution. The overall structure of this subunit is unique, demonstrating that archaeal type II enzymes are distinct from other type II topoisomerases. However, the core structure contains a pair of domains that are also found in type IA and classic type II topoisomerases. Together, these regions may form the basis of a DNA cleavage mechanism shared among these enzymes. The core A subunit is a dimer that contains a deep groove that spans both protomers. The dimer architecture suggests that DNA is bound in the groove, across the A subunit interface, and that the two monomers separate during DNA transport. The A subunit of topoisomerase VI is homologous to the meiotic recombination factor, Spo11, and this structure can serve as a template for probing Spo11 function in eukaryotes.

• Desogus G, Onesti S, Brick P, Rossi M, Pisani FM
• Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii.
• Nucleic Acids Res. 1999; 27: 4444-50
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• We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The analysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the small subunit of the eukaryotic DNA primase (the p50 subunit of the polymerase alpha-primase complex). This protein has been overexpressed in Escherichia coli and purified to near homogeneity. Recombinant Mjpri is able to synthesise oligoribonucleotides on various pyrimidine single-stranded DNA templates [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg(2+), Mn(2+)or Zn(2+), and is additionally stimulated by the monovalent cation K(+). A multiple sequence alignment has revealed that most of the regions that are conserved in eukaryotic p50 subunits are also present in the archaeal primases, including the conserved negatively charged residues, which have been shown to be essential for catalysis in the mouse primase. Of the four cysteine residues that have been postulated to make up a putative Zn-binding motif, two are not present in the archaeal homologue. This is the first report on the biochemical characterisation of an archaeal DNA primase.

• Zhu CX, Roche CJ, Papanicolaou N, DiPietrantonio A, Tse-Dinh YC
• Site-directed mutagenesis of conserved aspartates, glutamates and arginines in the active site region of Escherichia coli DNA topoisomerase I.
• J Biol Chem. 1998; 273: 8783-9
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• To catalyze relaxation of supercoiled DNA, DNA topoisomerases form a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl group on the DNA phosphodiester backbone bond during the step of DNA cleavage. Strand passage then takes place to change the linking number. This is followed by DNA religation during which the displaced DNA hydroxyl group attacks the phosphotyrosine linkage to reform the DNA phosphodiester bond. Mg(II) is required for the relaxation activity of type IA and type II DNA topoisomerases. A number of conserved amino acids with acidic and basic side chains are present near Tyr-319 in the active site of the crystal structure of the 67-kDa N-terminal fragment of Escherichia coli DNA topoisomerase I. Their roles in enzyme catalysis were investigated by site-directed mutation to alanine. Mutation of Arg-136 abolished all the enzyme relaxation activity even though DNA cleavage activity was retained. The Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 mutants had partial loss of relaxation activity in vitro. All the mutants failed to complement chromosomal topA mutation in E. coli AS17 at 42 degreesC, possibly accounting for the conservation of these residues in evolution.

• Chen SJ, Wang JC
• Identification of active site residues in Escherichia coli DNA topoisomerase I.
• J Biol Chem. 1998; 273: 6050-6
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• Alanine substitution mutagenesis of Escherichia coli DNA topoisomerase I, a member of the type IA subfamily of DNA topoisomerases, was carried out to identify amino acid side chains that are involved in transesterification between DNA and the active site tyrosine Tyr-319 of the enzyme. Twelve polar residues that are highly conserved among the type IA enzymes, Glu-9, His-33, Asp-111, Glu-115, Gln-309, Glu-313, Thr-318, Arg-321, Thr-322, Asp-323, His-365, and Thr-496, were selected for alanine substitution. Each of the mutant enzymes was overexpressed, purified, and characterized. Surprisingly, only substitution at Glu-9 and Arg-321 was found to reduce the DNA relaxation activity of the enzyme to an insignificant level. The R321A mutant enzyme, but not the E9A mutant enzyme, was found to retain a reduced level of DNA cleavage activity. Two additional mutant enzymes R321K and E9Q were also constructed and purified. Replacing Arg-321 by lysine has little effect on enzymatic activities; replacing Glu-9 by glutamine greatly reduces the supercoil removal activity but not the DNA cleavage and rejoining activities. From these results and the locations of the amino acids in the crystal structure of the enzyme, it appears that Glu-9 has a critical role in DNA breakage and rejoining, probably through its interaction with the 3' deoxyribosyl oxygen. The positively charged Arg-321 may also participate in these reactions by interacting with the scissile DNA phosphate as a monodentate. Because of the strict conservation of these residues, the findings for the E. coli enzyme are likely to apply to all type IA DNA topoisomerases.

• Berger JM
• Structure of DNA topoisomerases.
• Biochim Biophys Acta. 1998; 1400: 3-18
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• Over the last several years topoisomerases have finally begun to yield to high-resolution structural studies. These models have greatly aided our understanding of the mechanisms of topoisomerase catalysis and drug interactions. This review will cover advances in the structural biology of topoisomerases and discuss their implications for topoisomerase function.

• Berger JM, Fass D, Wang JC, Harrison SC
• Structural similarities between topoisomerases that cleave one or both DNA strands.
• Proc Natl Acad Sci U S A. 1998; 95: 7876-81
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• Type IA and type II DNA topoisomerases are distinguished by their ability to cleave one or two strands, respectively, of a DNA duplex. Both types have been proposed to use an "enzyme-bridging" mechanism, in which a break is formed in a DNA strand and a gap is opened between the broken pieces to allow passage of a second DNA strand or duplex segment. Although the type IA and type II topoisomerase structures appear overall quite different from one another, unexpected similarities between several structural elements suggest that members of the two subfamilies may use comparable mechanisms to bind and cleave DNA.

• Shuman S
• Vaccinia virus DNA topoisomerase: a model eukaryotic type IB enzyme.
• Biochim Biophys Acta. 1998; 1400: 321-37
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• Vaccinia topoisomerase has proven to be an instructive model system for mechanistic studies of the type IB family of DNA topoisomerases. The catalytically relevant functional groups at the active site and the circumferential topoisomerase-DNA interface were correctly surmised by mutational and footprint analysis of vaccinia topoisomerase in advance of structure determinations by X-ray crystallography. It is now evident from multiple crystal structures that the catalytic domains of type IB topoisomerases and site specific recombinases derive from a common ancestral strand transferase capable of forming a DNA-(3'-phosphotyrosyl)-enzyme intermediate. A constellation of conserved amino acids catalyzes attack of the tyrosine nucleophile on the scissile phosphate. Domain dynamics and DNA-induced conformational changes within the catalytic domain are likely to play a role in triggering strand scission and coordinating the strand exchange or strand passage steps.

• Kato S, Kikuchi A
• DNA topoisomerase: the key enzyme that regulates DNA super structure.
• Nagoya J Med Sci. 1998; 61: 11-26
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• DNA is a macromolecule carrying all genetic information and it must be packed in the cell nucleus. DNA is a double helix where one strand coils around the other. The helix is unwound and rewound every now and then to take out its information, which requires local alteration of the helical structure, resulting in the super-coiling of DNA. For replication, all the coils must be unwound at least once, and two daughter molecules are often catenated to each other. To solve these problems caused by the helical structure of DNA, topoisomerase activity introducing the transient breaking and rejoining of DNA strand is essential to perform each DNA transaction. In this article, we first review the mechanistic aspect of topoisomerase activity, and then discuss its basic clinical importance.

• Janin J
• Proteins with a ring.
• Structure. 1994; 2: 571-3
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• Proteins come in all sizes and shapes. Those which fold into a ring with a large hole in the middle may act as a clamp on DNA, a polysaccharide or another protein.

• Pansegrau W, Lanka E
• A common sequence motif among prokaryotic DNA primases.
• Nucleic Acids Res. 1992; 20: 4931-4931