The conformation of constitutive DNA interaction sites for eukaryotic DNA topoisomerase I on intrinsically curved DNAs

Bioc~hnica et Biophysica Acta, 1129 (1991 ) 73-82

73

© I tjgl Elsevier Science Publishers B.V. All rights reserved 0167-4781/91/$03.50

BBAEXP 92316

The conformation of constitutive D N A interaction sites for eukaryotic D N A topoisomerase I on intrinsically curved DNAs Giorgio Camilloni !, Micaela Caserta i, Andrea Amadei -" and Ernesto Di Mauro ~'-~ ! Centro AcMi Nucleich CNR, c /o Diparthnento di Genetica e Biolologia Molecolare, Unicersit~ di Roma 'La Sapien:a', Rome (Italy) and 2 Diparthnemo di Genetica e Biologia Molecolare, Unit~,'sit~ di Roma 'La S~q~ienza : Rome (L'al.v)

(Received 21 March 1991) (Revised manuscript received I August 1991)

Key words: DNA topoisomcrase I: DNA-proteir~ interactian: DNA conformation

The analysis of the sites which are cleaved constitutively and preferentially by eukaryotic DNA topoisomerase 1 on two intrinsically curved DNAs reveals the conformational features that provoke the cleavage reaction on the curve-inducing sequence elements in the absence of supercoiling. This analysis is based on the observation (Caserta et al. (1989) Nucleic Acids Res. 17, 8521-8532 and (1990) Biochemistry 29, 8152-8157) that the reaction of eukaryotic DNA topoisomerase 1 occurs on two types of DNA sites: sites S (Supercoiled induced) and sites C (Constitutive, whose presence is topology-independent). We report that sites C are abundant on the intrinsically curved DNAs analyzed. The DNAs studied were two intrinsically curved segments of different origin: the Crithidia fa~iculata kinetoplast DNA and the bent.containing domain B of the Saccharomyces cerevisiae ARSI. On these DNA segments DNA topoisomerase 1 cleaves at the junctions between the poly(A) tracts and mixed-sequence DNA. Analysis of the conformation of the double helix around the cleavage sites has revealed that the reaction occurs in correspondence of a defined DNA conformational motif. This motif is described by the set of Eulerian angular values that define the axial path of DNA (helical twist, deflection angle, direction) and of the orthogonal components of wedge (roll and tilt).

Introduce.on

The site-specificity of proteins whose function is related to DNA topology is in many instances determined by the bendability of DNA. In the paradigmatic example, the nucleosomes, histone octamers bind specifically to various DNA sequences bearing no apparent similarity. In this case it has been shown that site selection depends on the periodic arraagement of short sequence motifs which are more easi~iy bendable [12,23,28]. The DNA-protein systems in which DNA bending appears to be the driving force or a major determinant of specific interaction have been recently discm:.sed [29-31]. The use of the conformationai properties of DNA as recognition signals solves the problem of site selection among infinitely variable combina-

Correspondence: E. Di Mauro, Dipartimento di Genetica e Biologia Molecolare, Universit.~ di Roma 'La Sapienza', P. le Aldo Moro 5. 00185 Rome. Italy.

tions of sequences by proteins whose ~opology-related function demands non-random action or positioning b u t whose interaction sites are spread all over the genome. DNA topoisomerase i is one important instance of this type of proteins. Previous analyses have described the relationship between DNA topology and eukaryotic DNA topoisomerase I: this enzyme utilizes as substrate both positively and negatively supercoiled DNAs [34,4,22]; the rate of cleavage and of consequent relaxation directely correlates with the superhelical density [4,5]; perfectly relaxed DNA is not topoisomerized [4,5]. Neither the catalytic rate constants of the nicking-closing reactio:l nor the binding constaat increase as a function of the torsional strain [7], showing that the topolegy-dependent step of the topoisomerization reaction is the reactivi~ty of competent DNA sites [6,7]. The high binding affinity of DNA topoisomerase I for DNA [7] favors the hypothesis that eukaryotic DNA topoisomerase 1 is a resident component of chromatin. The increase of topological strain, both negative or positive, acti,~ates

74 potentially cleavable DNA sites, defined as S (Supercoiled induced) sites [6,7], Evidence for the existance of C (= constitutive) sites (i.e., DNA sites which are not topology-dependent and are reactive also on relaxed and linear DNAs) has been obtained in intrinsically bent DNAs [6], We have ana!yzed the distribution of the cleavages induced by calf thymus, chicken erythrocyte, wheat germ and Saccharomyces ceret,isiae DNA topoisomerase 1 on the particularly reactive constitutive DNA sites present on curved DNAs. The DNAs studied were the 211 bp kinetoplast curved DNA segment from C fasciculata [20] and on the curved domain B of the S. cerevtdae TRPI autonomously replicating sequence (ARSI) [35,21]. We report data obtained for the calf thymus enzyme (the enzymes from the other sources produced similar cleavage patterns - not shown). The results show that on both DNAs cleavages occur at the DNA sites which cause DNA curvature; we define below the conformational consensus for DNA topoisomerase I on these sites. Materials and Methods

Chicken erythrocyte DNA topoisomerase I was purified according to Ref. 21. The purified enzyme had a specific activity of 4.10 -~ U/rag; units are defined as the amount of enzyme that relaxes completely 1 ~tg of pBR322 DNA in 30 rain at 37 °C in low salt buffer [15]. Wheat germ and calf thymus DNA topoisomerases ! were purchased respectively from Promega Biotech, Madison, Wl and New England Biolabs. S. cerecisiae DNA topoisomerase l was purified in this laboratory by R. Negri from a proteinase-minus strain according to Ref. 14 and it had a specific activity of 10 4 U/rag. DNA modi~ing enzymes were from New England Biolabs and Boehringer, radiochemicals from New England Nuclear. DNAs used are: (1) a 265 bp DNA segment composed by the pSP65 polylinker from EcoR! to HindIIl, encompassing the C. fasciculata 211 bp Stul-Accl segment [20] cloned in Bam Hi sites. Linear DNAs were labeled at the 3' extremity by standard Klenow reaction. Secondary restriction was at the polylinker sites Sma ! (for analysis of the A strand) or Xbal (for the T strand). (2) The 237 bp DNA segment (from HindIII to Bglll) functionally defined as domain B of the S. ceret'isiae ARSI [35]. 3' labeling as for DNA #1. Secondary restriction: A&! (for the A strand), Pstl (for the T strand). Reaction of DNA fragments with DNA topoiso. merase ! and localization of cleavage sites was performed as described ia Ref. 5 and as detailed in the legends to Figs. 1 and 3. DNA curvature refers to the non-linear trajectory of the helix axis due to certain sequence arrangements,

DNA bending rt:.fers to the change of the helix axis imposed by protein bending. Results

Cleacage sites by calf thymus DNA topoisomerase i on C. fasciculata curt,ed DNA Localization of cleal,age sites Fig. 1 shows the localization of the cleavage sites on both the A and the T strand (A and B, respectively), in the absence of camptothecin, the A strand is efficiently cleaved at 14 sites (Fig. IA, lane 1). These sites almost exclusively localize at positions immediately distal to the T residue present at the 3' extremity of the runs of As (see Fig. IC). Only one minor site localizes outside the extremity of the runs of As (site #5). In the presence of camptothecin (this drug allows the accumulation of the cleaved intermediates of the topoisomerization reaction) the complexity of the pattern increases and minor cleavage sites become evident (Fig. 2A, lane 1). In conclusion, the sequence motif 'runs of A + IT', is not a mandatory requirement for the occurrence of preferential cleavage (i.e., the cleavage in the absence of camptothecin); there are 'As + T' which are not cut and cuts which are not on 'As + T' (see also the analysis of the ARSI sequence reported below), but this motif is highly preferentially cleaved. On the T strand, only one site (#18) is evident over the background in the absence of camptothecin (Fig. IB lane 1). Minor cleavage sites become evident upon induction by the drug (Fig. 2B lane 1); they occur almost exclusively in proximity of T residues and do not correlate with the curve-generating sequences (see map in Fig. IC). Conform~ tionai requirements The ~ lalysis of the sequences preferentially cleaved by DNA topoisomerase 1 provides several informations on the conformational requirements of this enzyme. DNA topoisomerase 1 does not require melted or easely melting sequences. Comparison with micrococcal nuclease. The cleavage pattern produced by DNA topoisomerase l on the A strand is very similar to the cleavage pattern by micrococcal nuclease (not shown). This latter enzyme recognizes both single-strand exposure and the identity of an unpaired base (A or T) [11]. Therefore, site-selection by DNA topoisomcrase I could appear to be associated with facilitated local exposure, presumably of a T (preference by DNA topoisomerase I for Ts has been reported [13,1]). However, the ApT step (present in every 'run of As + 1T' site, immediately before the cleavage position) is not an easely melting step. In support, the ApGs (an easily melting step) present in the sequence are not prefer-

75 entially cleaved. Thus, D N A topoisomerase 1 does not recognize melted or easely melting structures.

Analysis of the Eulerian .components of axial DNA

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path and of the orthogo, al compo.ents of the wedge of the sequences whic'h surromtd cleat age sites. The analysis reported below describes the conformational properties of the D N A tracts that allow preferential interaction with D N A topoisomerase I. An algorithm that defines how the axial path of D N A can be described at each step by three Eulerian angles (the helical twist, the deflection angle (wedge angle), and the direction of the deflection), has been recently reported [2]. The same algorithm defines the orthogonai components of wedge (roll and tilt). The sequence dependent variations of the five parameters (helical twist, wedge angle, direction of the deflection, roll and tilt)in the C..fascictdata DNA segment were analyzed according to this algorithm: the results are reported in Fig. 3. The purpose of this analysis is the identification of a conformational consensus for the cleavage sites on the sequences that induce the curvature on the DNA axis. The common features that are revealed by this analysis are:

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Fig. I. Cleavages by calf thymus DNA topoi.,,omerase ! on the 211 bp bent DNA. (A) The 'runs of As' .~n'and (A strand). Lane l. 5 U of DNA topo ! were reacted [5] fl)r 5 rain with 10 ng of linear 3' labeled 265 bp DNA. Secondary restriction: Sinai, Lane 2. 2 ng of pancreatic DNase ! [!1]. 30 s "A+G" indicates a standard A + G Maxam and Gilbert sequencing reaction on the same DNA fragment. Accurate mapping of the upper sites was oblained ~ith longer electrophoretic runs of the same samples (not .,,ilown). (B) As in A for the T strand. Secondary. restriction: Xha!. L,me !. topo h lane 2. DNase !. Reactions in the absence of camptothecin. Cleavage sites arc numbered. Control reaction without enzymatic treatmeilt showed minor spontaneous breakage at one position (brackets in ia,e:,; 1)(not detailed). Correction of 1115 map po~ition of sites #1 and 2 according to Rcf. 13. (C') Mapping of cleavage sites in the absence of camptothecin (filled triangles). I. the presence of canlptothecin, additional minor cleavage sites are detected on the T strand (open triangles). Camptothecin-induced sites are from the experiment reported in Fig. 2. Arrows: DNase i cleavage sites.

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Fig, 2, The effect of distamycin on the cleavage reaction. Reactions as in Fig. I, in the presence of 10 ~,M camptothecin. (A) A strand in the absence (lane I) and in the presence of 0.1, 0,2, 0.3, 0.5 and I p.M distamycin (from 2 to 6), 5 U of DNA topoisomerase I. (B) Same for the T strand,

(i) A defined pattern of twist angles (the 'V'-shaped motif indicated by the thick line at the 5' side of the cleaved positions) (Fig. 3A and B, first panel from the top).

(ii) A series of continuous wedge angles (indicated by the shaded areas in the second panel). (iii) The homogeneity of the direction of these wedge angles. The direction is negative (third panel, shaded areas). (iv) A defined pattern of roll angles (the 'reverseV'-shaped motif indicated by the thick line at the 5' side of the cleaved positions (fourth panel). (v) No common features are observed for the tilt angle. (vi) No common features are observed for any of the parameters analyzed at the positions 3' of the cleaved sites. Distamycbz. Distamycin changes the local DNA conformation which results in curvature [16,17]. We observe that distamycin also abolishes the preferential cleavage by DNA topoisomerase ! at the extremity of A-tracts (Fig. 2, sites #1 to 13 in A), but does not abolish cleavage at the other positions. The cleavage pattern obtained in the presence of low concentration of distamycin (Fig. 2) shows that at 1/zM all the 'run of As + IT' cleavage sites in the A-strand are abolished (A). On the T strand (B), the site at the extremity of the AATT block (#18) is inhibited, the other two major sites (present in the non-bending positions, #13 and #23) are not. These sites are actually enhanced by the conformational variation induced on the DNA by the drug (Fig. 2B). This set of data indicates that the local deformation of the double helix which induces curvature is related to the set of conformational parameters recognized by eukaryotic DNA topoisomerases l. DNase !. The interaction of DNase I with DNA is sequence independent but is strongly inhibited by deformations of the minor groove [11]; the relevance of appropriate local conformation for cleavage has been shown [26]. The pattern of DNase I cleavage sites on the C. fasciculata DNA was determined: the DNase I sensitive positions are periodically distributed over both strands (with the same periodicity observed for the curvature-inducing sequences) and in many positions the cuts face each other cross the strands with the regular 2-4 bp cross-strand stagger described (Fig. IA, lane 2, B, lane 2, and C). The DNase I sites never map on the runs of As and alternate with the DNA topoisomerase I sites. The alternation of the cleavage patterns produced by the two enzymes further supports the conclusion of their opposite conformational require-

Fig, 3, Determination of the Eulerian components of the axial DNA path and of the orthogonal components of the wedge angle for the C.

fuscictdata curved tract, (A) DNA sequence from position I to position I00. (B) DNA sequence from position I01 to 223. From top to bottom: the values of helical twist, the deflection (wedge) angle, the direction of the deflection, the roll angle, the tilt angle. Calculations are based on the •,'alues reported by Bolshoy et al. [2], The positions of the sites cleaved by DNA topoisomerase I (numbered from # I to 14) are reported. Tile common features observed are indicated by thickening of the line (twist and roll) and by shaded areas (wedge and direction of deflection).

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78 quence is reported in B. Also in this case, distamycin preferentially inhibits cleavages on the curvature-sites (namely, sites #3, 4, 5 and 6) (not shown). Analysis of the Eulerian and orthogonal angles around the cut positions was performed as described above for the C fasciculata DNA (Fig. 5).

ments: undeformed helical parameters for DNase ! [11], a local deformation for DNA topoisomerase 1.

Cleavage sites in the domain B of S. ceret,isiae ARSI The results obtained for the C. fasciculata bent DNA are confirmed by a similar analysis performed on the bent-containing segment B of the S. cerevisiae ARSI. Fig. 4 shows the cleavage sites on both the A and ~he T strands of this DNA (lanes 1 and 2, respectively). Localization of the cleavage sites on the se-

Discussion We have shown that cleavage by eukaryotic DNA topoisomerases I on curved DNAs occurs at the sites

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Fig. 4. Cleavages on the 237 bp segment of S. ceret'isiae ARSI. (A) Reaction with DNA topoi as reported in Fig. 1. 100/tM camptothecin. In the absence of this drug the same sites were observed (not shown). (B) Localization of the cleaved sites on the sequence. The bend-inducing stretches of As are underlined.

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24~Hnp nosLtlon Fig. 5. Determination of the Eulerian components of the axial DNA path and of the orthogonal component of the wedge angle for the S. cerevisiae ARSI DNA. Analysis performed as reported in Fig. 3. The numbering reported (from 1 to 242) is referred to a complete DNA segment (i.e., a segment with uncleaved terminal restriction sites).

80

s

that induce curvature. Cleavages occur distally to the T residue present at the 3' extremity of the runs of adenines. The requirement for a T residue at 5' of the cleavage by DNA topoisomerase I has been reported previously [13,1]. The preferential cleavage (i.e., cleavage in the absence of camptothecin) in the C fasciculata curved DNA occurs at DNA sites which are characterized by a defined set of conformational properties. These properties have been analyzed using the recently published algorithm for the determination of the Eulerian angles of the DNA axial path and of the orthogonal components of the wedge [2]. The ¢onformational consensus of the preferential cleavage sites is the following: (i) Common features at the 5' side of the ~:leaved sites; absence of common features at the 3' side:. (ii) The common features at 5' are: (a) a 'V'-shaped set of twist angles; (b) a series of negatively-d~rected deflection (wedge) angles; (c) a 'reverse-V'-shaped set of roll angles; and (d) no common motif for tilt ~ngles. in addition, the following should be noted: (i) all the A-tracts, being of a common sequence type - AAAAT or AAAAAT - could be expecled to behave similarly t~ward DNA topoisomerase !. At the contrary, the intensity of the various cleavage si~.es is markedly different: a hierarchy can be established by scanning densitometry (not shown) of the cleavage pattern: site #4 > #6 > #2 > #10 > #12, etc. One site predicted on the basis of the DNA sequence alone is absent from the actual cleavage pattern (at base pair #21 on the numbering system reported in Fig. IC). These facts clearly indicate a sequence context-effect for DNA topoisomerase ! reactivity. (ii) The absence of any consensus for the tilt angle values around the cleavage sites reveals the irrelevance of this parameter for the DNA topoisomerase i reactivity. This fact could not have been predicted on the basis of the sequence alone. The en~me cleaves several other sites on the 'A strand' of the two curved DNA segments analyzed. These sites are localized outside the curvature-inducing tracts: site #5 at the C. fasciculata A strand, site #1 (a minor site) and sites # 7 - 9 on the ARSI A-strand. The conformation of the sequences surrounding these sites shows no correlation with the conformational consensus described for the cleavage sites that localize on the curvature-inducing tracts (not shown). The conformation of these sites is encompassed in the complex catalogue of DNA topoisomerase ! sites described by $hen and Shen [24] (see below).

Related et,idences An analysis of the specificity of the recognition of DNA helical structure by eukaryotic DNA topoisomerase I has been reported [24], which describes the

specific structural features of hundreds of camp tothecm-induced cleavage sites. The study that we pre. sent here differs from the work of Shen and Sher because we analyze the specific class of sites which have the following properties: they are curvaturespecific, highly reactive, cleaved constitutively (i.e., not supercoiling induced) and they can be revealed even in the ab~nce of camptothecin. Alteration of the helix twist angles and of the roll angles in the sequences which encompass both camptothecin-induced cleavages and the cleavage in the l~;~,h!y ~w,~f¢.~.ntia! 16 bp sequence [27] has been observed [18]. Stabilization by helix curvature of DNA topoisomerase I-DNA interaction on a high affinity binding site and mutant derivatives thereof has been reported [19].

A role for ctm'ed DNA In addition to the informations provided on DNAenzyme recognition mechanisms, our finding of the preferential cleavage on the curvature-inducing DNA sequences and their conformational characterization has interest per se, because it defines an interactive property of curved DNA. The curvature of DNA in the absence of external constraints results from the repetition of special sequence or structural motifs (generally runs of adenines) in phase with the DNA helical repeat. Since its discovery [32,20], the nature of the structural properties of the double-strand that cause bending has been the object of intense and sometimes controversial investigations (for reviews, see Refs. 33, 28, 10 and 30). Curvature caused by sequence elements other than AA has also been described [21. A recent review [9] offers a unifying model of the curvature inducing structures and points to the fact that all models that seek to explain DNA bending require a difference in base pair inclination between the A-tracts that cause bending and the intervening B-DNA structures.

Base pairs inclination and groot'e width We note at this point that the sites cleaved by DNA topoisomerase ! on the curvature-inducing sequences are located at the junctions between the segments (A-tracts and B-DNA) characterized by such different base pair inclinations [9]. in addition, it has been shown that in A-tracts the width of the minor groove decreases smoothly from the 5 ' ~ 3 ' end of the A-tract, as measured by the frequency of backbone cleavage by hydroxyl radical [3]. At the end of the A tract the groove width returns to normal values, as shown also by the localization of the

81 DNAse 1 cuts (see section 'Conformational requirements' and Ref. 11). Theretore, the cleavage sites that we have described at the end of A-tracts occur in a position that can be considered as a sort of junction between sequences characterized by different basepair inclination [9] and different groove width [3]. Whenever a T residue is present at these junctions DNA topoisomerase 1 cleaves. In fact, cleavage occurs on 19 out of 20 AAAAT or AAAAAT tracts analyzed (15 in Crithidia, 4 in the S. ceret'isiae ARS I).

DNA bendability The periodic arrangement of short sequence motifs which are more easely bendable provides a recognition signal for nucleosomes [28,29] and several other proteins (reviewed in Ref. 31). DNA which is actually and intrinsically bent (and not only potentially bendable) in the correct direction is a highly preferential substrate for interaction with aucleosomes [8]. As for DNA topoisomerase !, ,:mr data indicate that a single AAAAT motif is sufficient for the activation of cleavage. This indication derives from the observation that site #14 in the Crithidia DNA is separated from next clevage site (#13) by 53 base pairs (see Fig. IC) and can therefore be considered as an isolated cleaved site. However, this type of evidence is not conclusive in defining whether the preferential reaction by DNA topoisomerase I on curved DNA is caused by the overall curvature of the DNA molecule or by a local conformation. The fact that it has been possible to define a con/'ormational consensus for preferential cleavage sites (i.e., a defined alteration of the twist angle, a negative deflection angle, a defined alteration of the roll angle) argues that DNA topoisomerase ! recognizes a local, precisely defined DNA conformation and not an actual curvature of the whole molecule or its potential bendability. Curved DNA sequences have been observed in functionally strategic locations (centromeres, telomers, origins of ~:eplication, promoters, enhancers, gyrase sites). These are all positions where genetic processes may accumulate topological strain. We have previously provided evidence for a topological regulation of the DNA topoisomerase I reaction (see Introduction). Both cleavage and topoisomerization are activated by topological strain and are inhibited by relaxation of DNA. Evidence has been provided [5] that the variation of the writhing of the double strand is the relevant topological component in this activation process. This is supported by the fact that intrinsically curved DNA is a preferential substrate for DNA topoisomerase I [6]. The interaction of DNA topoisomerase 1 with DNA sites whose conformation we have determined in this

study might serve the important biological role of releasing the tension which accumulates in processes such as trancription [36].

Acknowledgments This work was supported by Fondazione 'PasteurFondazione Cenci Bolognetti' (Universith di RomaL by Programma Finalizzato Biotecnologie (CNR) and by P.F. ingegneria Genetica. We thank G. Micheli and R. Perini for developping the computer program and for the help in the elaboration of the data. C fasciculata DNA was kindly provided by P.T. Englund.

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