a type ib topoisomerase with dna repair activities
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A type IB topoisomerase with DNA repair activitiesGalina I. Belova*, Rajendra Prasad, Sergei A. Kozyavkin, James A. Lake, Samuel H. Wilson and Alexei I. Slesarev*i
*Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117871, Russia; Laboratory of Structural Biology,National Institute on Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709; Fidelity Systems, Inc.,Gaithersburg, MD 20879; and Molecular Biology Institute, University of California, Los Angeles, CA 90095
Edited by James C. Wang, Harvard University, Cambridge, MA, and approved March 16, 2001 (received for review January 24, 2001 )
Previously we have characterized type IB DNA topoisomerase V(topo V) in the hyperthermophile Methanopyrus kandleri. Theenzyme has a powerful topoisomerase activity and is abundant inM. kandleri. Here we report two characterizations of topo V. First,we found that its N-terminal domain has sequence homology withboth eukaryotic type IB topoisomerases and the integrase family oftyrosine recombinases. The C-terminal part of the sequence in-cludes 12 repeats, each repeat consisting of two similar but distincthelix-hairpin-helix motifs; the same arrangement is seen in recom-bination protein RuvA and mammalian DNA polymerase b. Second,on the basis of sequence homology between topo V and polymer-ase b, we predict and demonstrate that topo V possesses apurinicyapyrimidinic (AP) site-processing activities that are important inbase excision DNA repair: (i) it incises the phosphodiester backboneat the AP site, and (ii) at the AP endonuclease cleaved AP site, itremoves the 5* 2-deoxyribose 5-phosphate moiety so that a single-nucleotide gap with a 3*-hydroxyl and 5*-phosphate can be filledby a DNA polymerase. Topo V is thus the prototype for a newsubfamily of type IB topoisomerases and is the first example of atopoisomerase with associated DNA repair activities.
A ll DNA topoisomerases are classified into types I and II,according to the nature of a transient break in DNA, singleor double stranded, respectively (1). Type I enzymes are furtherclassified into two families on the basis of their biochemicalproperties: type IA enzymes form a transient covalent bond tothe 59 phosphoryl of the broken strand, whereas type IB enzymesform a 39-end covalent bond. Members of the same family sharesignificant sequence similarities and are structurally related(25). The type IA family of topoisomerases, which includestopoisomerases I and III (68) and thermophilic reverse gyrases(912), is widespread in nature and not confined to prokaryotessolely. Until recently, type IB enzymes have been positivelyidentified only in eukaryotes. We have previously purified andcharacterized type I DNA topoisomerase V (topo V) from thehyperthermophile Methanopyrus kandleri (1315). The enzymehas the following properties in common with eukaryotic topo-isomerase I, which distinguish it from all other known prokary-otic topoisomerases: (i) its activity is Mg21-independent; (ii) itrelaxes both positively and negatively supercoiled DNA; and (iii)it makes a covalent complex with the 39 end of the broken DNAstrand. The discovery of topo V suggests that type IB enzymesmay also be ubiquitous and not confined to eukaryotes, asoriginally thought. We have now cloned and sequenced its geneby using peptidic sequence information. The finding of unex-pected sequence homology with enzymes involved in DNArepair prompted us to examine whether topo V itself might haveDNA repair activities.
Materials and MethodsN-Terminal Sequencing of Topo V and Development of DNA Probes.Sequencing of the N termini of topo V was performed on anApplied Biosystems 471A protein sequencer. Five hundredpicomols of topo V (14) was digested with Endoproteinase Lys-Cat 37C for 1 h in buffer containing 25 mM TriszHCl (pH 8.0) and400 mM NaCl. The proteolytic fragments were separated bySDSyPAGE, blotted to Immobilon-P transfer membrane (Mil-lipore), and subjected to 45 cycles of Edman degradation. The
N-terminal sequence analysis of topo V and its N-terminalfragments was found to be ALVYDAEFVGSEREFEEERET-FLKGVKAYDGVLATRYLMERSSSA. The second N-terminal sequence was derived from a 35-kDa polypeptide andwas determined to be KSGRQERSEEEWKEWLERKVGE-GRARRLIEYFGSA. The alanine in the first position indicatesthat the N terminus was processed.
The flanking seven amino acid stretches of the 45- and 35-aasequences were used to prepare degenerate oligonucleotideprimers. These primers were used in PCR to generate 135- and105-bp nucleotide probes. The probes were then cloned andsequenced to confirm their usefulness.
Cloning and Sequencing of the top5 Gene. Genomic DNA of M.kandleri and the l library were prepared as described (16). Twouniformly labeled PCR probes were prepared by using theQuickPrime kit (Amersham Pharmacia) and (a-32P) dCTP(NEN). With these probes, about 2,000 plaques were screened.Five l clones hybridizing to both probes were isolated. The top5gene was cloned on a 6.5-kb EcoRIyPstI fragment. This fragmentwas subcloned into pBluescript II SK(1), generating pAS65, andthen used as a template for DNA sequencing. The problems insequencing of guaninecytosine-rich (60%) Methanopyrus DNAwere resolved by the addition of ThermoFidelase (FidelitySystems, Gaithersburg, MD) to an AmpliCycle kit (PerkinElmer). The topo V sequence within the cloned 6.5-kb EcoRIyPstI genomic fragment was identified by comparing the trans-lation product with the N-terminal 45- and internal 35-aasequences derived from protein-sequencing experiments.
Recombinant Proteins. The top5 gene and the gene fragmentcovering amino acids 1532 were amplified by PCR from pAS65and cloned into pET21b or pET14 vectors (Novagen), generatingpET21TV and pET14T61 expression plasmids, which encodefull-size topo V and its 61-kDa N-terminal fragments, respec-tively. Proteins were overexpressed in Escherichia coliBL21(DE3) pLysS cells (Novagen) and purified essentially asdescribed (14).
Mutagenesis. The tyrosine residues were replaced with phenyl-alanine in the top5 gene by using a site-directed mutagenesis kit(QuickChange, Stratagene) on pET21bTV and pET14T61. Af-ter mutagenesis, the presence of desired nucleotide changes wasverified by DNA sequencing. Mutant proteins were isolated asdescribed above.
Topoisomerase, ApurinicyApyrimidinic (AP) Lyase, and 2-Deoxyribose5-Phosphate (dRP) Lyase Assays. The topoisomerase activity wasdetermined as described (13), except that the truncated topo V
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: HhH, helix-hairpin-helix; HTH, helix-turn-helix; dRP, 2-deoxyribose 5-phos-phate; AP, apurinic/apyrimidinic; topo V, topoisomerase V; b-pol, polymerase b.
Data deposition: The sequence reported in this paper has been deposited in the GenBankdatabase (accession no. AF311944).
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www.pnas.orgycgiydoiy10.1073ypnas.111040498 PNAS u May 22, 2001 u vol. 98 u no. 11 u 60156020
proteins were assayed in low salt (0100 mM NaCl). AP lyaseand dRP lyase assays were performed essentially as described(1719). Briefly, the reaction mixture for the AP lyase activity oftopo V consisted of 20 nM 32P-labeled (39) duplex DNA sub-strate containing an AP site at position 21 (top strand, 59-AGCTACCATGCCTGCACGAAUTAAGCAATTCGTA-ATCATGGTCATAGCT-39), 50 mM Hepes (pH 7.5), 1 mMEDTA, and 0.2 M NaCl. The assay was performed at 60C for5 min. The reaction products were stabilized by addition ofNaBH4 (340 mM) and then separated by electrophoresis in a20% polyacrylamide gel (7 M urea, 90 mM Trisy64.6 mM boricacidy2.5 mM EDTA, pH 8.3). Gels were dried and visualized byautoradiography. To quantify the product, gels were scanned ona Molecular Dynamics PhosphorImager model 450, and the datawere analyzed by using IMAGEQUANT software. The dRP lyaseactivity was assayed under similar reaction conditions as for APlyase, except that the uracil-DNA glycosylase-reacted DNAsubstrate was further treated with AP endonuclease in thepresence of 5 mM MgCl2 to generate an incised AP-DNA.
Trapping of topo VDNA covalent complex by alkali orNaBH4 was performed as described previously (13, 19).
Gel Retardation Assay. Reaction mixture containing 200 nM topoV, 20 nM 32P-labeled substrates (double-stranded oligonucleo-tides containing uracil, AP, or dRP sites at position 21), 50 mMHepes, pH 7.5, 0.2 M NaCl was incubated for 5 min at 37C. Anequal volume of 23 Triszborate EDTA buffer with 20% glycerolwas added to the binding reaction, and the samples wereanalyzed by 6% PAGE. The extent of binding was visualized byautoradiography.
Results and DiscussionCloning and Sequence Analysis of TOP5. Topo V has been cloned ona 6.5-kb EcoRIyPstI genomic fragment, which was identified by
screening of a M. kandleri l library with two PCR probes derivedfrom N-terminal and internal protein sequences of topo V.Sequence analysis of topo V revealed an ORF encoding a proteinof 988 amino acids, with a predicted molecular mass of 112 kDa.This is in a very good agreement with the molecular mass of 110kDa deduced from SDSyPAGE (2). The coding sequence agreeswith the protein-sequencing data, although there are 5 codonsfor RVKVV directly 59 of the codon for the first alanine. Thetranslation starting point of the top5 gene has not yet beenidentified. On the basis of our sequencing data, we assume thatthe initiator codon is the first GTG (valine) preceding GCG(alanine). The lack of a good