MolecularstructureofnickedDNA:A substrate for DNA ... · Proc. Nati. Acad. Sci. USA87(1990) 2527 A4 T4 FIG. 2. Stereoscopic difference Fourierelectron density mapat the position ofthe

Proc. Natl. Acad. Sci. USAVol. 87, pp. 2526-2530, April 1990Biochemistry

Molecular structure of nicked DNA: A substrate for DNArepair enzymes

(x-ray diffraction/DNA conformation)

JUAN AYMAMI*t, MIQUEL COLL*t, Gus A. VAN DER MAREL§, JACQUES H. VAN BooM§,ANDREW H.-J. WANG¶, AND ALEXANDER RICH**Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; §Gorlaeus Laboratories, Leiden State University, Leiden, 2300RA, TheNetherlands; and IDepartment of Physiology and Biophysics, University of Illinois at Urbana-Champaign, Urbana, IL 61801

Contributed by Alexander Rich, January 5, 1990

ABSTRACT The molecular structure of a nicked dodeca-mer DNA double helix, made of a ternary system containingd(CGCGAAAACGCG) + d(CGCGTT) + d(TTCGCG) oligo-nucleotides, has been determined by x-ray diffraction analysisat 3 A resolution. The molecule adopts a B-DNA conformation,not unlike those found in intact dodecamer DNA moleculescrystallized in a somewhat different crystal lattice, despite agap due to the absence of a phosphate group in the molecule.The helix has a distinct narrow minor groove near the centerof the molecule at the AAAA region. This suggests that theinternal stabilizing forces due to base stacking and hydrogen-bonding interactions are sufficient to overcome the loss ofconnectivity associated with the disruption of the covalentbackbone of DNA.

DNA is known to be damaged in a number of ways andbiological systems have developed a variety of enzymes torepair different types ofDNA damage (1). A common DNAdamage is the limited hydrolysis of a sugar-phosphate bondfollowed by the removal of the phosphate group. This pro-duces a nickedDNA duplex in which only one strand ofDNAis continuous while its opposite strand is interrupted by themissing phosphate group. One of the intriguing questionsassociated with this type of damages is its consequence onDNA conformation. For example, does the double helixretain its structural integrity at the nicked site? How does therepair enzyme recognize the structural anomaly, ifany, ofthedouble helix? We have approached this problem by solvingthe three-dimensional structure of such a nicked DNA com-posed of a single strand of 12 nucleotides d(CGCGAAAT-TCGCG) bound to two complementary smallerDNA strands,each a hexanucleoside pentaphosphate [d(CGCGTT) + d(TT-CGCG)]. These three oligonucleotide strands form a DNAdodecamer duplex with a structure not unlike that of otherrelated B-DNA dodecamers (2-8), despite the fact that thenicked duplex crystallizes in a different crystal lattice.Around the nicked site, the 3' hydroxyl group of the lastthymidine residue in one hexamer forms a hydrogen bondwith the 5' hydroxyl group of the first thymidine residue ofthe following hexamer, bridging the two hexamer strands atthe site where the phosphate group is missing. This moleculecrystallizes in the lattice with a twofold orientational disorderwith about equal populations in each orientation.

EXPERIMENTALThe oligonucleotides were synthesized on a Pharmacia DNAsynthesizer and purified by HPLC. A typical crystallizationmixture contained each oligonucleotide at 0.5 mM, 2.9 mM

FIG. 1. Schematic drawings of the two related orthorhombiccrystal packing arrangements of the DNA dodecamers. (A) d(CGC-GATATCGCG) + netropsin complex (6). (B) d(CGC-GAAAACGCG) + d(CGCGTT) + d(TTCGCG). The b axis ishorizontal and the c axis is vertical. The gap in the black band of thehelix represents the location of the missing phosphate.

MgCl2, 29 mM sodium cacodylate (pH 5), 8.6 mM sper-mine-4HC1, and 1.9 mM Hoechst Pharmaceuticals 33258 dyeand 2% in 2-methyl-2,4-pentanediol (2-MPD). The solutionwas equilibrated with 50% 2-MPD at 4TC. Crystals appearedafter a week, but they were too small for diffraction study. Afew small but clean crystals were chosen for seeding exper-iments. One crystal grew to the dimensions 0.1 X 0.3 X 1.0mm after repeated seedings and it was used for data collec-tion. The Hoechst 33258 dye was included in the crystalli-zation solution since it was believed that it might stabilizeduplex formation by binding in the minor groove of B-DNAat the central four A-T base pairs. While the Hoechst mole-cule was used for crystal growth, no dye molecule was foundin the crystal after the refinement. The crystals were in thespace group P212121 with unit cell dimensions a = 25.99, b =44.03, and c = 66.62 A. The crystal was mounted in a sealedglass capillary with a droplet of mother liquor for datacollection on a Rigaku AFC-5R rotating anode x-ray diffrac-tometer at 250C using the c-scan mode with CuKa radiation.Data were collected to a resolution of =3.0 A; 553 reflectionswere observed at the 2.0 ao(F) level above the background andwere used in the refinement. Each of the three individualmolecules was also tested for crystallization, but no crystalwas obtained. This suggested that the duplex in the crystalarose from the association of all three DNA strands, instead

tPresent address: Unidad de Quimica Macromolecular, E.T.C. In-ziniers Industrials, Diagonal 647, 08028 Barcelona, Spain.tPresent address: Max-Planck Institut fur Biochemie, 8033 Martins-ried bei Munchen, F.R.G.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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A4

T4

FIG. 2. Stereoscopic difference Fourier electron density map at the position of the missing phosphate in both A4 and T4 models.

of other unlikely combinations such as a dodecamer duplexwith four AA mismatches.Although the unit cell dimensions resemble those found in

other DNA dodecamer crystals (a 25.7, b 41.3, and c66.9 A) (2-8), the volume is =4o0 greater than in the otherdodecamer crystals, mostly because of a longer b axis. Thediffraction pattern suggested that this structure was notisomorphous to other related dodecamer crystals. The struc-ture was solved by the molecular replacement method usingthe program ULTIMA (9) with a B-DNA model derived fromthe molecular structure of a DNA dodecamer. The newcrystal lattice had a different packing with the moleculeundergoing a rotation of ==50° and a translation of _4 A alongthe c axis relative to other dodecamer crystals (2-8) as shownin Fig. 1. This resulted in the loss of hydrogen bonds that areused in stabilizing the original dodecamer lattice in the adirection. However, in the new crystal lattice the interactionsalong the c axis are retained with the same G-G hydrogenbonds involving the terminal two COG base pairs in the minorgrooves between adjacent dodecamer molecules related bythe twofold screw axis (2, 10).The structure was refined by using the Konnert-

Hendrickson constrained refinement procedure (11). Duringthe refinement, it became evident that the molecule occupiedthe same position in two orientations, related by its pseudo-molecular twofold axis perpendicular to the helix axis. This isillustrated in Fig. 2, which compares the final differenceFourier electron density maps of two molecular models re-sulting from these orientations. They are denoted A4 and T4

models since one model has A4 in one strand, while the othermodel is gapped and has T2 + T2 in the same position. It canbe seen that in bothA4 andT4 models there is residual electrondensity at the gap position arising from the missing phosphategroup between the two hexamers. Both were independentlyrefined in the same manner. The A4 model was refined to anR factor of 18.2% with a root mean square (rms) difference ofbond distances of 0.017 A and it contains 79 water molecules.In the T4 model, the corresponding finalR factor is 18.6% withthe rms differences in bond distances of 0.016 A including 67water molecules. The A4 and T4 models had a rms differenceof 1.32 A and 1.38 A, respectively, in their atomic positionswhen compared with the DNA molecule in the netropsin-d(CGCGATATCGCG) complex (6). The rms deviations fromthe structure of other dodecamer molecules were calculatedwithout using the central A-T base pairs.

In view of the limited number of diffraction reflections, wedid not attempt to refine the structure by using a model witha weighted sum of the two orientations (A4 + T4 model). Itshould be pointed out that a disorder similar to this has beenseen for the d(CGCAAAAATCGC + CGCATl'Tl'GCG)duplex (12) and in the netropsin-d(CGCGATATCGCG) com-plex in the original dodecamer orthorhombic crystal lattice(6).11

lThe atomic coordinates and structure factors will be deposited inthe Protein Data Bank, Chemistry Department, Brookhaven Na-tional Laboratory, Upton, NY 11973.

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RESULTS AND DISCUSSIONThe present structure has been determined at only mediumresolution; nonetheless, a number of interesting features canbe seen. First, the molecule is clearly in the B-DNA confor-mation. This is shown in the stereoscopic (2FO-FC) Fouriermap with the T4 model fitted within its electron density (Fig.3). Fig. 4 depicts the stereoscopic skeletal and van der Waalsmodels of the T4 model. In the skeletal model shown in Fig.4A, the nick in the sugar-phosphate backbone is indicated byan arrow. There is a hydrogen bond (2.9 A) between the 5'hydroxyl of one thymidine group with the 3' hydroxyl of theother, shown as a dashed line. The van der Waals diagram ofFig. 4B shows that the two oxygen atoms (0-5' and 0-3') arevirtually in contact with each other across the gap. Inaddition, as shown in Figs. 3 and 4A, some of the base pairsin the structure appear to be highly propeller-twisted, espe-cially near the nick in the center of the molecule. Althoughthe limited resolution of this crystal structure does not allowus to draw unambiguous conclusions about these details ofthe conformation, it would not be surprising that such dis-tortions in base stacking actually exist.A characteristic of B-DNA that has emerged from a

number of related dodecamer DNA crystal structure studiesis that the double helix has a narrow minor groove in theB-DNA double helix in a stretch containing several APT basepairs (2-8, 12). The phosphorus to phosphorus distanceacross the minor groove is in the range of 13.3-14.3 A in theG-C region, while this distance decreases to 9.3-10.8 A in theAPT regions. This narrowness of the minor groove in theA-T-rich regions provides the appropriate environment forthe binding site for many crescent-shaped planar moleculessuch as netropsin and distamycin (13, 14). The narrowing ofthe minor groove is found in this region independent ofwhether or not the groove binding molecules are present.However, there has been some question relating to whetherthe minor groove width is associated with a particular crystallattice.

The nicked dodecamer duplex in this structure shows asignificant narrowing of the minor groove surrounding theAT base pairs even though the continuity of the covalentbackbone in the middle of the T residues is no longer intact.Fig. 5 shows the cylindrical projection ofthe two double helixmodels of the nicked dodecamer DNA molecules. It can beseen that the 04' atom to 0-4' atom distances which measurethe minor groove width across the center of the moleculeencompassing the APT base pairs is 3-4 A narrower than thecomparable distances found in the G*C base pairs at the endofthe molecule. Furthermore, this narrow groove exists eventhough these molecules are packed in a different crystallattice from the previous dodecamer structures. This nickedstructure thus shows that the narrow minor groove in the APTregion is independent of the continuity of the sugar-phosphate backbone and it occurs with different crystallattice interactions. Thus, it is likely to be an intrinsicsequence-dependent property of the DNA molecule.

It should be noted that a narrowed A-T-tract minor groovewas also seen in the structure of a repressor-DNA complexwhere it could have been influenced by the protein-DNAinteractions (15). Furthermore, that complex contains DNAduplexes joined by a 1-base-pair overlap, producing a doublynicked DNA helix. A similar doubly nicked DNA conforma-tion is also found in a DNase I-octanucleotide complex (16).The present structure is the first ternary oligonucleotidecomplex of B-DNA free of bound protein. The generalconclusion reached by this study is that the cohesive internalforces stabilizing the DNA molecule, largely base stackingand hydrogen bonding, are great enough to overcome the lossof connectivity associated with disruption of the covalentbackbone ofDNA. On this score, it is interesting to note thatstacking interactions are the predominant forces stabilizingmany crystal structures of DNA oligonucleotides (10). Forexample, in the atomic resolution Z-DNA structure ofd(CGCGCG) hexamer crystals, the base-pair stacking pat-tern between the two end-on-end hexamers is almost exactlyidentical to that of the internal GpC step even though bothphosphates are missing across the helix (17).

FIG. 3. Stereoscopic drawing of the (2F - Fj) electron density map with the T4 dodecamer model fitted in the electron density envelopegenerated by the program FRODO (20).

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FIG. 4. (A) Skeletal diagram of the ternary dodecamer double helix showing the missing phosphate group between the two hexamers[d(CGCGTT) and d(TTCGCG)]. There appears to be a hydrogen bond between the 0-3' hydroxyl group from the d(CGCGTT) hexamer andthe 0-5' hydroxyl group of the d(TTCGCG) hexamer, which is shown as a dashed line (arrows). (B) The van der Waals view of the same nickeddodecamer duplex. The 0-3' and 0-5' groups mentioned in the text are almost touching each other. It can be seen that the minor groove is quitenarrow near the middle of the helix at the AAAA stretch. The gap is indicated with an arrow in both figures.

A related parallel study on the influence of a nick in thesugar-phosphate backbone on the stability ofthe double helixhas been carried out recently by nuclear magnetic spectros-copy. The result also suggested the existence of a duplex

structure for the ternary DNA molecules d(AGCCGTACT-GCA) + d(ACGGCT) + d(TGCAGT) in solution (18).

In conclusion, these results show that the major features ofthe B-DNA double helix can be maintained even ifone strand

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FIG. 5. Cylindrical projection of the nicked DNA dodecamer ofboth A4 and T4 models. The gap is marked by a large arrow. Theminor groove width is estimated by the 0-4'-0-4' distances acrossthe groove as shown by the numbers.

is severed through the loss of a phosphate group. Thus, inscanning the DNA double helix, DNA repair enzymes actingon such a structure must detect either small variations in theconformation of the double helix or, more probably, the lackofthe phosphate group itself. While the DNA structure in thecomplex of DNA-repair enzyme may be significantly differ-ent from that of the uncomplexed DNA molecule [as in thecase of DNA-EcoRI complex (19)], the present structure islikely the one that is seen initially by the repair enzyme.

This work was supported by grants from the National Institutes ofHealth and National Science Foundation (A.R. and A.H.-J.W.) and

from National Aeronautics and Space Administration, Office ofNaval Research, and the American Cancer Society (A.R.).G.A.v.d.M. and J.H.v.B. were supported by the Netherlands Orga-nization for the Advancement of Pure Research (ZWO). J.A. was aNorth Atlantic Treaty Organization Fellow. M.C. was a FulbrightFellow.

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