Individual Nucleotide Bases, Not Base Pairs, Are Critical for Triggering Site-specific DNA Cleavage by Vaccinia Topoisomerase
2004; Elsevier BV; Volume: 279; Issue: 38 Linguagem: Inglês
10.1074/jbc.m407376200
ISSN1083-351X
AutoresLigeng Tian, Jane M. Sayer, Donald M. Jerina, Stewart Shuman,
Tópico(s)Synthesis and Biological Evaluation
ResumoVaccinia DNA topoisomerase forms a covalent DNA-(3′-phosphotyrosyl)-enzyme intermediate at a specific target site 5′-C+5C+4C+3T+2T+1p↓N-1 in duplex DNA. Here we study the effects of abasic lesions at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. The rate of DNA incision was reduced by factors of 350, 250, 60, and 10 when abasic sites replaced the -1N, +1T, +2T, and +4C bases of the scissile strand, but abasic lesions at +5C and +3C had little or no effect. Abasic lesions in the nonscissile strand in lieu of +4G, +3G, +2A, and +1A reduced the rate of cleavage by factors of 130, 150, 10, and 5, whereas abasic lesions at +5G and -1N had no effect. The striking positional asymmetry of abasic interference on the scissile and nonscissile strands highlights the importance of individual bases, not base pairs, in promoting DNA cleavage. The rate of single-turnover DNA religation by the covalent topoisomerase-DNA complex was insensitive to abasic sites within the CCCTT sequence of the scissile strand, but an abasic lesion at the 5′-OH nucleoside (-1N) of the attacking DNA strand slowed the rate of religation by a factor of 600. Nonscissile strand abasic lesions at +1A and -1N slowed the rate of religation by factors of ∼140 and 20, respectively, and strongly skewed the cleavage-religation equilibrium toward the covalent complex. Thus, abasic lesions immediately flanking the cleavage site act as topoisomerase poisons. Vaccinia DNA topoisomerase forms a covalent DNA-(3′-phosphotyrosyl)-enzyme intermediate at a specific target site 5′-C+5C+4C+3T+2T+1p↓N-1 in duplex DNA. Here we study the effects of abasic lesions at individual positions of the scissile and nonscissile strands on the rate of single-turnover DNA transesterification and the cleavage-religation equilibrium. The rate of DNA incision was reduced by factors of 350, 250, 60, and 10 when abasic sites replaced the -1N, +1T, +2T, and +4C bases of the scissile strand, but abasic lesions at +5C and +3C had little or no effect. Abasic lesions in the nonscissile strand in lieu of +4G, +3G, +2A, and +1A reduced the rate of cleavage by factors of 130, 150, 10, and 5, whereas abasic lesions at +5G and -1N had no effect. The striking positional asymmetry of abasic interference on the scissile and nonscissile strands highlights the importance of individual bases, not base pairs, in promoting DNA cleavage. The rate of single-turnover DNA religation by the covalent topoisomerase-DNA complex was insensitive to abasic sites within the CCCTT sequence of the scissile strand, but an abasic lesion at the 5′-OH nucleoside (-1N) of the attacking DNA strand slowed the rate of religation by a factor of 600. Nonscissile strand abasic lesions at +1A and -1N slowed the rate of religation by factors of ∼140 and 20, respectively, and strongly skewed the cleavage-religation equilibrium toward the covalent complex. Thus, abasic lesions immediately flanking the cleavage site act as topoisomerase poisons. Poxvirus topoisomerases are exemplary type IB topoisomerase family members; they cleave and rejoin one strand of the DNA duplex through a transient DNA-(3′-phosphotyrosyl)-enzyme intermediate. Vaccinia topoisomerase cleaves duplex DNA at a pentapyrimidine target sequence, 5′-(T/C)CCTTp↓ (1Shuman S. Prescott J. J. Biol. Chem. 1990; 265: 17826-17836Abstract Full Text PDF PubMed Google Scholar). (The Tp↓ nucleotide is defined as the +1 nucleotide.) Topoisomerases encoded by other genera of poxviruses recognize the same DNA target sequence (2Hwang Y. Wang B. Bushman F.D. J. Virol. 1998; 72: 3401-3406Crossref PubMed Google Scholar, 3Klemperer N. Lyttle D.J. Tauzin D. Traktman P. Robinson A.J. Virology. 1995; 206: 203-215Crossref PubMed Scopus (27) Google Scholar, 4Palaniyar N. Fisher C. Parks R. Evans D.H. Virology. 1996; 221: 351-354Crossref PubMed Scopus (15) Google Scholar, 5Petersen B.Ø. Hall R.L. Moyer R.W. Shuman S. Virology. 1997; 230: 197-206Crossref PubMed Scopus (17) Google Scholar, 6Krogh B.O. Cheng C. Burgin A. Shuman S. Virology. 1999; 264: 441-451Crossref PubMed Scopus (18) Google Scholar), despite the large variations in overall G/C contents of the genomes of the different poxvirus genera. Available structural and biochemical studies suggest that the assembly of a catalytically competent topoisomerase active site is triggered by recognition of the 5′-CCCTT/3′-GGGAA target sequence (7Cheng C. Kussie P. Pavletich N. Shuman S. Cell. 1998; 92: 841-850Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 8Tian L. Claeboe C.D. Hecht S.M. Shuman S. Structure (Lond.). 2004; 12: 31-40Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Early studies using nuclease footprinting, modification interference, modification protection, analog substitution, and UV cross-linking techniques suggested that vaccinia topoisomerase makes contact with several nucleotide bases and the sugar-phosphate backbone of DNA within and immediately flanking the CCCTT element (9Shuman S. J. Biol. Chem. 1991; 266: 11372-11379Abstract Full Text PDF PubMed Google Scholar, 10Shuman S. Turner J. J. Biol. Chem. 1993; 268: 18943-18950Abstract Full Text PDF PubMed Google Scholar, 11Sekiguchi J. Shuman S. J. Biol. Chem. 1994; 269: 31731-31734Abstract Full Text PDF PubMed Google Scholar, 12Sekiguchi J. Shuman S. J. Biol. Chem. 1996; 271: 19436-19442Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 13Sekiguchi J. Shuman S. EMBO J. 1996; 15: 3448-3457Crossref PubMed Scopus (46) Google Scholar, 14Cheng C. Shuman S. Biochemistry. 1999; 38: 16599-16612Crossref PubMed Scopus (18) Google Scholar, 15Hwang Y. Burgin A. Bushman F. J. Biol. Chem. 1999; 274: 9160-9168Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Recent studies have focused on delineating the features of the DNA interface that affect the kinetics of transesterification. For example, position-specific covalent polycyclic aromatic hydrocarbon diol epoxide-DNA adducts have been exploited to probe the minor groove interface (16Tian L. Sayer J.M. Kroth H. Kalena G. Jerina D.M. Shuman S. J. Biol. Chem. 2003; 278: 9905-9911Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar) and the effects of intercalation at all of the dinucleotides steps spanning the target site (17Yakovleva L. Tian L. Sayer J.M. Kalena G.P. Kroth H. Jerina D.M. Shuman S. J. Biol. Chem. 2003; 278: 42170-42177Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 18Yakovleva L. Handy C.J. Sayer J.M. Pirrung M. Jerina D.M. Shuman S. J. Biol. Chem. 2004; 279: 23335-23342Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The aromatic hydrocarbon adduct studies delineated the margins of the functional DNA interface at atomic resolution but did not reveal the nature of the DNA contacts within the essential zone of DNA. Modifications of the nonbridging and 5′-bridging oxygens of the DNA phosphodiester backbone have been especially informative in that regard. Phosphorothioate and methylphosphonate modifications at the scissile phosphodiester have illuminated the chemical mechanism of topoisomerase IB, the roles of the individual amino acids in either transition state stabilization or general acid catalysis, and the parameters affecting the stability of the covalent topoisomerase-DNA intermediate (19Krogh B.O. Shuman S. Mol. Cell. 2000; 5: 1035-1041Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 20Stivers J.T. Jagadeesh G.J. Nawrot B. Stec W.J. Shuman S. Biochemistry. 2000; 39: 5561-5572Crossref PubMed Scopus (59) Google Scholar, 21Krogh B.O. Claeboe C.D. Hecht S.M. Shuman S. J. Biol. Chem. 2001; 276: 20907-20912Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 22Krogh B.O. Shuman S. J. Biol. Chem. 2002; 277: 5711-5714Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 23Tian L. Claeboe C.D. Hecht S.M. Shuman S. Mol. Cell. 2003; 12: 199-208Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Position-specific interference by methylphosphonate modifications at remote phosphates on the scissile and nonscissile strands has provided an atomic resolution map of the DNA backbone contacts required for active site assembly (8Tian L. Claeboe C.D. Hecht S.M. Shuman S. Structure (Lond.). 2004; 12: 31-40Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Whereas sterically subtle modifications of nonbridging phosphate oxygens flanking the cleavage site can have drastic effects on transesterification chemistry (8Tian L. Claeboe C.D. Hecht S.M. Shuman S. Structure (Lond.). 2004; 12: 31-40Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), phosphorothiolate substitutions for the 5′-bridging oxygens of the scissile strand have no significant effect on the rate of DNA cleavage by vaccinia topoisomerase (6Krogh B.O. Cheng C. Burgin A. Shuman S. Virology. 1999; 264: 441-451Crossref PubMed Scopus (18) Google Scholar). A major outstanding question is how the poxvirus topoisomerase reads the nucleotide sequence at its cleavage site. Available evidence suggests that most of the site specificity is achieved at the level of transesterification chemistry rather than at the noncovalent DNA binding step (24Sekiguchi J. Shuman S. Nucleic Acids Res. 1994; 22: 5360-5365Crossref PubMed Scopus (32) Google Scholar). Whereas the affinity for the target site, the rate of cleavage, and the cleavage equilibrium constant (Kcl) are affected by the nucleotide sequence context surrounding the 5′-(C/T)CCTT target site (1Shuman S. Prescott J. J. Biol. Chem. 1990; 265: 17826-17836Abstract Full Text PDF PubMed Google Scholar, 15Hwang Y. Burgin A. Bushman F. J. Biol. Chem. 1999; 274: 9160-9168Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) in ways that are not well understood in structural terms, the dominant factor triggering the DNA incision reaction is the pentamer 5′-CCCTT/3′-GGGAA. We have begun to systematically address the features of the individual bases that affect the kinetics of DNA cleavage via position-specific base modifications entailing relatively small additions to, or subtractions from, the standard base structures (11Sekiguchi J. Shuman S. J. Biol. Chem. 1994; 269: 31731-31734Abstract Full Text PDF PubMed Google Scholar, 17Yakovleva L. Tian L. Sayer J.M. Kalena G.P. Kroth H. Jerina D.M. Shuman S. J. Biol. Chem. 2003; 278: 42170-42177Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), as well as modification by more bulky adducts (16Tian L. Sayer J.M. Kroth H. Kalena G. Jerina D.M. Shuman S. J. Biol. Chem. 2003; 278: 9905-9911Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 18Yakovleva L. Handy C.J. Sayer J.M. Pirrung M. Jerina D.M. Shuman S. J. Biol. Chem. 2004; 279: 23335-23342Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The addition of new substituents to the purine and pyrimidine rings provides a means of mapping functionally relevant sites of protein-DNA contact. The caveat to the new substituent approach is that a particular site of modification interference (presumably arising via steric hindrance) cannot be equated with a specific atomic interaction with the DNA. Rather, the power of the new substituent approach resides mainly in its ability to identify DNA structural elements that are not functionally relevant. Moreover, the sensitivity of the base modification method necessarily depends on the extent to which the particular modification alters the size, shape, and hydrogen-bonding potential of the base or base pair. In general, the most informative modification interference effects will be those elicited by the subtlest modifications. A corollary of this proposition is that the most straightforward approach to assessing the relevance of a given base to topoisomerase catalysis is to remove the base rather than add new substituents to the base. Missing base analysis has been facilitated by the availability of synthetic DNAs containing position-specific tetrahydrofuran (THF) 1The abbreviation used is: THF, tetrahydrofuran. abasic sites (Fig. 1). Other investigators have shown that abasic lesions flanking cleavage sites for mammalian DNA topoisomerase I or topoisomerase II can act as “topoisomerase poisons” that trap the normally transient covalent topoisomerase-DNA intermediate by causing a selective slowing of the religation transesterification reaction relative to the cleavage transesterification reaction (25Pourquier P. Ueng L. Kohlhagen G. Mazumder A. Gupta M. Kohn K.W. Pommier Y. J. Biol. Chem. 1997; 272: 7792-7796Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 26Kingma P.S. Osheroff N. J. Biol. Chem. 1997; 272: 1148-1155Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Such studies have broad physiological relevance insofar as: (i) abasic lesions arise in vivo with high frequency as a consequence of base-excision repair by DNA glycosidases, and (ii) topoisomerases may reinforce the cytotoxicity of DNA lesions (27Nitiss J.L. Nitiss K.C. Rose A. Waltman J.L. J. Biol. Chem. 2001; 276: 26708-26714Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Here we conduct a systematic analysis of the effects of abasic lesions at six positions of the scissile strand and six positions of the nonscissile strand within the target sequence 5′-CCCTTA/3′-GGGAAT. The key instructive findings are that individual bases, not the base pairs, are critical determinants of the rate of DNA cleavage. We also find that abasic lesions at the +1 and -1 positions flanking the scissile phosphodiester slow the rate of religation and thereby poison the topoisomerase reaction equilibrium. DNA Substrates—Oligodeoxyriboncucleotides containing single THF abasic sites were commercially synthesized and gel-purified by Oligos Etc. Inc. (Wilsonville, OR) or synthesized by standard solid-phase methodology and purified by reverse-phase high performance liquid chromatography before and after removal of the 5′-dimethoxytrityl protecting group. The CCCTT-containing scissile strands were 5′-32P-labeled by enzymatic phosphorylation in the presence of [γ-32P]ATP and T4 polynucleotide kinase. The labeled oligonucleotides were gel-purified and hybridized to standard or modified nonscissile strand oligonucleotides at a 1:4 molar ratio of scissile to nonscissile strand. Annealing reaction mixtures containing 0.2 m NaCl and oligonucleotides were heated to 80 °C and then slow-cooled to 22 °C. The hybridized DNAs were stored at 4 °C. The structures of the annealed duplexes and the sequences of the component strands are depicted in the figures. Vaccinia Topoisomerase—Recombinant vaccinia topoisomerase was produced in Escherichia coli (BL21) by infection with bacteriophage λCE6 (28Shuman S. Golder M. Moss B. J. Biol. Chem. 1988; 263: 16401-16407Abstract Full Text PDF PubMed Google Scholar) and then purified to apparent homogeneity from the soluble bacterial lysate by phosphocellulose and Source S-15 chromatography steps. Protein concentration was determined by using the dye binding method (Bio-Rad) with bovine serum albumin as the standard. Single-turnover DNA Cleavage—Reaction mixtures containing (per 20 μl) 50 mm Tris-HCl (pH 7.5), 0.3 pmol of CCCTT-containing DNA, and 75, 150, or 300 ng (2, 4, or 8 pmol) of vaccinia topoisomerase were incubated at 37 °C. Aliquots (20 μl) were withdrawn at various times and quenched immediately with SDS (1% final concentration). The products were analyzed by electrophoresis through a 10% polyacrylamide gel containing 0.1% SDS. Free DNA migrated near the dye front. Covalent complex formation was revealed by transfer of radiolabeled DNA to the topoisomerase. The extent of covalent complex formation was quantified by scanning the dried gel using a Fujifilm BAS-2500 imager. A plot of the percentage of input DNA cleaved versus time established the end point values for cleavage. The data were then normalized to the end point values (defined as 100%), and the cleavage rate constants (kcl) were calculated by fitting the normalized data to the equation 100 - % cleavage(norm) = 100e-kt. Single-turnover Religation—Cleavage reaction mixtures containing (per 20 μl) 0.3 pmol of 32P-labeled 18-mer/30-mer DNA (unmodified or abasic) and 2, 4, or 8 pmol of topoisomerase were incubated at 37 °C for 10-60 min to form the suicide intermediate. Religation was initiated by the simultaneous addition of NaCl to 0.5 m anda5′-OH 18-mer acceptor strand d(GTTCCGATAGTGACTACA) to a concentration of 15 pmol/22 μl (i.e. a 50-fold molar excess over the input DNA substrate). Aliquots were withdrawn at various times and quenched immediately with 1% SDS. A time 0 sample was withdrawn prior to addition of the acceptor strand. The samples were digested for 60 min at 37 °C with 10 μg of proteinase K, then mixed with an equal volume of 95% formamide 20 mm EDTA, heat-denatured, and analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 m urea in TBE (90 mm Tris borate, 2.5 mm EDTA). Religation of the covalently bound 12-mer strand to the 18-mer acceptor DNA yielded a 5′-32P-labeled 30-mer strand transfer product. The extent of religation (expressed as the percent of the covalent intermediate converted into 30-mer) was plotted as a function of reaction time. The data were normalized to the end point values and krel was determined by fitting the data to the equation 100 - % religated(norm) = 100e-kt. Equilibrium Cleavage—A 34-mer CCCTT-containing oligonucleotide was 5′-32P-labeled, then gel-purified and annealed to an unlabeled complementary 30-mer strand to form a duplex containing 12 bp of DNA 5′ to the cleavage site and 18 bp 3′ to the cleavage site. Reaction mixtures containing (per 20 μl) 50 mm Tris-HCl (pH 7.5), 0.3 pmol of 34-mer/30-mer DNA, and 9, 18, 37, 75, 150, or 300 ng of topoisomerase were incubated at 37 °C for 10 min. The reactions were initiated by the addition of topoisomerase to prewarmed reaction mixtures. The reaction was quenched by adding SDS to 0.5%. The samples were digested for 60 min at 37 °C with 10 μg of proteinase K, mixed with an equal volume of formamide/EDTA, and then analyzed by electrophoresis through a 17% polyacrylamide gel containing 7 m urea in TBE. The cleavage product, a 32P-labeled 12-mer bound to a short peptide, was well resolved from the input 34-mer substrate. The extent of strand cleavage was quantified by scanning the gel. The Kcl is defined as the ratio of covalently bound DNA to noncovalently bound DNA at the reaction end point under conditions of saturating enzyme and was calculated according to the equation Kcl = % cleaved/(100 - % cleaved). Position-specific Scissile Strand Abasic Interference Effects on DNA Cleavage—A series of oligodeoxynucleotide 18-mer scissile strands containing a single THF abasic site within the 5′-C+5C+4C+3T+2T+1A-1 sequence were 5′-32P-labeled and then annealed to an unlabeled 30-mer strand to form “suicide” cleavage substrates for vaccinia topoisomerase (Fig. 1B). The presence of the abasic lesions at the correct positions was assessed by limited digestion of the suicide substrates with E. coli exonuclease III, which possesses 3′-exonuclease and abasic endonuclease activities. Whereas exonuclease III digests the unmodified suicide substrate exclusively in the 3′-exonuclease mode to generate a ladder of partially shortened 5′-32P-labeled products (21Krogh B.O. Claeboe C.D. Hecht S.M. Shuman S. J. Biol. Chem. 2001; 276: 20907-20912Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), the abasic suicide substrates were also cleaved endonucleolytically at the 5′-phosphodiester flanking the abasic nucleoside to yield a discrete 5′-32P-labeled product (Fig. 2). Digestion in parallel of the six position-specific abasic substrates yielded a series of endonucleolytic cleavage products of the expected size and differing by 1-nucleotide spacing. The cleavage transesterification reaction of vaccinia topoisomerase results in covalent attachment of the 32P-labeled 12-mer 5′-pCGTGTCGCCCTTp to the enzyme via Tyr274. The unlabeled 6-mer 5′-OH leaving strand ATTCCC dissociates spontaneously from the protein-DNA complex. Loss of the leaving strand drives the reaction toward the covalent state, so that the reaction can be treated kinetically as a first-order unidirectional process. The reaction of excess topoisomerase with the unmodified control substrate attained an end point at which 95% of the DNA was converted to covalent topoisomerase-DNA complex; the reaction was complete within 20 s. The extent of transesterification after 5 s was 89% of the end point value. From this datum, we calculated a single-turnover kcl of 0.46 s-1 (Fig. 1B). Single abasic lesions spanning positions +5 to -1 on the scissile strand had no significant effect on the extents of DNA cleavage (90-98%), but they exerted disparate position-specific effects on the rate of the reaction. The kcl values for the +5 abasic (0.25 s-1) and +3 abasic substrates (0.18 s-1) were within a factor of 2 or 3 of the value for unmodified control DNA. In contrast, abasic lesions at +2, +1, and -1 slowed kcl by factors of 60 (to 0.0073 s-1), 260 (0.0018 s-1), and 350 (0.0013 s-1), respectively. The +4 abasic lesion caused a 10-fold decrement in kcl (0.043 s-1). These results indicate that: (i) vaccinia topoisomerase does not rely on contacts to the +5C or +3C bases during the forward transesterification reaction, and (ii) the most important contributions of the scissile strand bases are made by +2T, +1T, and -1N. Nonscissile Strand Abasic Effects on DNA Cleavage—An unmodified 5′-32P-labeled 18-mer scissile strand was annealed to a series of 30-mer nonscissile strands containing a single THF abasic site within the 3′-G+5G+4G+3A+2A+1T-1 element (Fig. 3). Single abasic lesions spanning positions +5 to -1 on the nonscissile strand had no significant effect on the extents of DNA cleavage (80-97%), but they elicited position-specific effects on the rate of cleavage that, with the exception of the +5 abasic site, were drastically different from the effects exerted by the loss of the complementary base on the scissile strand. The first instructive finding was that elimination of the +5G base at the “upstream” margin of the target site had no significant impact on kcl (0.33 s-1). Thus, neither component base of the +5C:G base pair was functionally important for the forward cleavage reaction. A second notable finding was that loss of the -1T base at the “downstream” margin on the nonscissile strand also had no effect on kcl (0.46 s-1), but in this case, there was a huge disparity between the benign effect of an abasic lesion on the nonscissile strand compared with the 350-fold decrement in kcl that occurred when the complementary -1A base was missing from the scissile strand. The kcl values for the +1 abasic (0.085 s-1), +2 abasic (0.046 s-1), +3 abasic (0.003 s-1), and +4 abasic (0.0035 s-1) substrates were slowed by factors of 5, 10, 150, and 130 relative to the kcl for unmodified DNA. A gradient of increasing severity of abasic interference was evident as the nonscissile strand lesion was phased away from the cleavage site; this contrasts with a severity gradient of opposite directionality for cleavage interference by abasic lesions on the scissile strand. The strand selectivity can be quantified as the ratio of the cleavage rate constants for the missing scissile strand and nonscissile strand bases of each base pair. These SS/NS abasic ratios are as follows: 0.75 for +5C:G, 12 for +4C:G, 60 for +3C:G, 0.15 for +2T:A, 0.02 for +1T:A, and 0.003 for -1A:T. Effects of Missing Base Pairs on DNA Cleavage—5′-32P-Labeled 18-mer scissile strands containing single abasic sites were annealed to abasic 30-mer nonscissile strands to form a series of suicide cleavage substrates lacking both complementary bases of each base pair within the CCCTTA element (Fig. 4). The effects of missing base pairs ranged from modest (e.g. +5, +3, and -1) to severe (+4, +2, and +1). The missing base pair interference effects we classify as modest were those that had little effect on the extent of cleavage (78-98%) and for which the rate decrement incurred by deleting both bases was either no worse than, or only modestly worse than, the interference caused by a single abasic lesion. We can quantify the missing base pair effect as the inverse ratio of the cleavage rate constant for the missing pair substrate to the slower of the two rate constants for a single abasic substrate lacking either the scissile or nonscissile strand base. For example, the missing base pair effect at position -1A:T was 1.1 (= 0.0013/0.0011), which means that complete elimination of the -1 base pair was no worse than deleting just the -1A base on the scissile strand. The missing base pair effect at +5C:G was 4 (= 0.25/0.059), but the notable finding was that the rate of cleavage of a DNA substrate lacking the +5C:G pair was slowed by only a factor of 8 compared with an unmodified DNA. The missing pair effect at +3C:G was 7 (= 0.003/0.00043). The +4, +2, and +1 missing pair interference effects we classify as severe affected the cleavage end point (11-62%) and elicited strongly synergistic effects on kcl compared with the single abasic lesions (Fig. 4). The missing base pair effects at +4C:G, +2T:A, and +1T:A were 440, 1300, and 240, respectively. It is likely that these severe effects of deleting both bases of the pair are caused by secondary structural changes to the DNA target site, especially to the phosphodiester backbone with which the topoisomerase makes electrostatic contacts that are essential for the cleavage reaction (8Tian L. Claeboe C.D. Hecht S.M. Shuman S. Structure (Lond.). 2004; 12: 31-40Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Position-specific Abasic Interference with DNA Religation—The observed abasic interference effects on the rate of DNA cleavage could reflect a requirement for specific bases for either: (i) chemical catalysis of transesterification or (ii) DNA-assisted assembly of a catalytically competent active site. The observed rate of covalent complex formation by vaccinia topoisomerase with unmodified DNA is believed to be limited by the chemical step itself rather than by requisite precleavage conformational steps (29Kwon K. Stivers J.T. J. Biol. Chem. 2002; 277: 345-352Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). The topoisomerase catalytic cycle entails two transesterification reactions, cleavage and religation. Religation occurs via the attack of the DNA 5′-OH on the covalent intermediate, leading to expulsion of the Tyr274 leaving group and restoration of the DNA phosphodiester backbone (30Shuman S. J. Biol. Chem. 1992; 267: 8620-8627Abstract Full Text PDF PubMed Google Scholar). Religation is believed to be the microscopic reversal of the cleavage reaction. Thus, changes in the structure of the topoisomerase or the DNA target site that inhibit the chemical step directly will likely slow both the forward cleavage reaction and the DNA religation reaction. However, changes that impede active site assembly to the point that it becomes limiting for cleavage will affect the cleavage reaction selectively; they would have less impact on the rate of religation by the preformed covalent topoisomerase-DNA intermediate, in which the active site is already assembled correctly. Abasic effects on the religation reaction were studied under single-turnover conditions by assaying the ability of preformed suicide intermediate to transfer the covalently held 5′-32P-labeled 12-mer strand to a 5′-OH-terminated 18-mer strand to generate a 30-mer product (Fig. 5A). After forming the suicide intermediate on the unmodified 18-mer/30-mer DNA substrate or 18-mer/30-mer DNA containing an abasic lesion at positions +5, +4, +3, +2, or +1 of the scissile strand, the religation reaction was initiated by adding a 50-fold molar excess of the 18-mer DNA acceptor strand. The sequence of the added 18-mer is fully complementary to the 5′ single-stranded tail of the suicide intermediate. The ionic strength was adjusted simultaneously to 0.5 m NaCl to promote dissociation of the topoisomerase after strand ligation and prevent recleavage of the 30-mer strand transfer product. Aliquots were withdrawn immediately prior to the addition of 18-mer and NaCl (defined as time 0) and at various times afterward, and the extent of religation at each time point was expressed as the fraction of the 32P-labeled DNA present as covalent adduct at time 0 that was converted to 30-mer strand transfer product (Fig. 5B). Religation by topoisomerase bound covalently on unmodified DNA was effectively complete within 5 s, the earliest time point analyzed. The religation of covalent complexes containing +5, +4, or +3 abasic sites on the scissile strand was virtually indistinguishable from that of unmodified DNA, as gauged by the completeness of the reactions after 5 s (Fig. 5B). The religation results are consistent with the minimal/mild effects of the +5, +4, and +3 abasic lesions on the rate of the forward cleavage reaction. Note that the religation rate constant of vaccinia topoisomerase (krel ∼1.0-1.2 s-1) is too fast to measure manually, which means that the religation rate would have to be slowed at least severalfold to be detectable in our assays. The religation of covalent complexes containing +2 or +1 abasic lesions was effectively complete in 10 s; the reactions attained 56 and 52% of the end point values after 5 s, from which we estimated krel values of 0.16 and 0.15 s-1, respectively. We surmise that the +2 and +1T bases contribute no more than a ∼6-fold enhancement of the rate of religation, which contrasts with their 60- and 250-fo
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