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The Crystal Structures of DNA Holliday Junctions P Shing Ho* and Brandt F Eichman†

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The Crystal Structures of DNA Holliday Junctions P Shing Ho* and Brandt F Eichman† 302 The crystal structures of DNA Holliday junctions P Shing Ho* and Brandt F Eichman† Nearly 40 years ago, Holliday proposed a four-stranded Molecular models of the four-way junction complex or junction as the central intermediate in the general It is now generally accepted that there are two structural mechanism of genetic recombination. During the past two forms of the Holliday junction (reviewed in [9•]). Low salt years, six single-crystal structures of such DNA junctions have conditions favor an open-X form, in which the four arms been determined by three different research groups. These are extended in a square planar geometry (Figure 1b), structures all essentially adopt the antiparallel stacked-X thereby minimizing the repulsion between the negatively conformation, but can be classified into three distinct charged phosphates concentrated at the junction. This is categories: RNA–DNA junctions; ACC trinucleotide junctions; also the form observed in complexes with the enzymes and drug-induced junctions. Together, these structures provide RuvA [10,11,12•] and Cre [13]. insight into how local and distant interactions help to define the detailed and general physical features of Holliday junctions In contrast, the presence of multivalent cations effective- at the atomic level. ly shields these negative charges, allowing the arms to coaxially pair and stack into the more compact stacked-X Addresses junction (Figure 1c). Before the recent crystal studies, *Department of Biochemistry and Biophysics, ALS 2011, Oregon the most detailed models of four-way junctions were State University, Corvallis, Oregon 97331, USA; derived from studies using gel electrophoresis, fluores- e-mail: [email protected] cence resonance energy transfer, NMR spectroscopy and †Department of Biological Chemistry and Molecular Pharmacology, • Harvard Medical School, Boston, Massachusetts 02115, USA atomic force microscopy [9 ]. These models showed that the stacked duplex arms across the junction of the Current Opinion in Structural Biology 2001, 11:302–308 antiparallel stacked-X conformation are related in a right- 0959-440X/01/$ — see front matter handed sense by an angle of approximately 60° © 2001 Elsevier Science Ltd. All rights reserved. [14,15,16•]. Furthermore, there are two distinct stacked- Abbreviations X conformational isomers (or conformers), depending on bp base pairs which DNA strands are exchanged between duplexes HMT 4′-hydroxymethyl-4,5′,8-trimethylpsoralen across the junction [17–19]. Lh-X left-handed stacked-X NER nucleotide excision repair Molecular models of the stacked-X junction have been Rh-X right-handed stacked-X constructed by simply redirecting the phosphodiester link- ages of two adjacent duplexes from theoretical models Introduction [20,21] or from crystal structures of resolved DNA double DNA recombination was first recognized as a means to helices [22–25] to generate four-stranded assemblies. The introduce genetic diversity in cells. More recently, the lattices of certain B-DNA crystals place the grooves of mechanism of recombination has become implicated as adjacent duplexes in close proximity and properly oriented an important cellular mechanism to repair (reviewed in to model a junction that maintains the base pairing [1]) or replicate through (reviewed in [2•]) DNA lesions. between nucleotides. NMR studies have supported the All of these processes are thought to undergo a mecha- general features of these models, showing that the base nism analogous to that proposed by Holliday [3] in 1964 pairs stacked across the junction are similar to those of for homologous recombination, which involves a four- B-DNA [18,26,27]. Although useful in describing general stranded intermediate [3–8] in which DNA strands structural features, these molecular models cannot eluci- cross-over between two homologous duplexes to effect an date the details of the Holliday junction at the atomic level exchange of genetic material (Figure 1a). The central role in the manner that single-crystal structures can. of the four-stranded Holliday junction in recombination has lead to numerous studies to characterize its physical Three classes of junction in crystal structures properties and innumerable attempts to crystallize Since the mid-1980s, there have been sporadic reports of the structure. In the past two years, four-way junctions various laboratories having crystallized a DNA Holliday have been seen in the crystal structures of DNA com- junction. Unfortunately, none of these resulted in a pub- plexes with two different proteins and of six nucleic lished structure. It is ironic that the structures of the first acid-only constructs. In this review, we will focus on nucleic acid junction [28••], the first DNA junction [29••] the intrinsic non-protein-dependent structures of the and the first junction in a homologous DNA sequence junction. Although none of these constructs was original- [30••] were all published within 12 months of each other ly designed to study junctions, our collective good and none was designed to study four-way junctions. fortune now provides us with the most detailed models Currently, there are six available crystal structures of DNA of Holliday junctions to date and insights into how they containing junctions, which fall into three classes: are stabilized. RNA–DNA junctions (Figure 2a); ACC trinucleotide The crystal structures of DNA Holliday junctions Ho and Eichman 303 Figure 1 Recombination and the Holliday junction. (a) (b) 5' 3' (a) The model for homologous recombination proposed by Holliday [3]. (b) The open-X form 5' A D of the Holliday junction, as seen in the 5' 3' 5' complex with the junction-resolving enzyme 5' 5' 3' Cre [13]. (c) The antiparallel Rh-X junction, as 5' B C seen in the ACC-type crystal structures. Nick 3' 5' Strand invasion and ligation Holliday junction Branch migration (c) 5' 3' 5' 3' 2 D 1 1 A C B 2 Strands Resolve Strands 3' 5' 3' 5' cut at 1 cut at 2 strands Patched Spliced Current Opinion in Structural Biology DNA junctions (Figure 2b); and psoralen (drug) cross- The two most recently determined structures reveal DNAs linked junctions (Figure 2c). that are cross-linked by 4′-hydroxymethyl-4,5′,8-trimethyl- psoralen (HMT) [32••]. The junction in HMT– The first RNA–DNA complex was designed originally to d(CCGGTACCGG) is a consequence of the stabilizing ACC study a DNA construct with catalytic RNAse activity (a trinucleotide in the DNA sequence, whereas the junction in DNAzyme), but it crystallized as a right-handed stacked-X HMT–d(CCGCTAGCGG) is induced by the drug — the junction [28••]. The most recent variation of this class of unmodified DNA crystallizes as resolved duplexes [30••]. junction is an RNA–DNA junction with the stacked The psoralen-bound structures are examples of the compet- duplexes related in a left-handed sense; this has been ing stacked-X conformers. The HMT–d(CCGGTACCGG) termed the crossed conformation [31••]. However, as the junction places the four-base-pair arms stacked over the six- two structures differ only in the handedness of the interdu- base-pair arms, with the furan-linked strands exchanging plex angle, in this review we will refer to them as the right- across the junction. Alternatively, the HMT–d(CCGC- handed (Rh-X) and the left-handed stacked-X (Lh-X) TAGCGG) junction has a six-over-four stacking arrangement RNA–DNA junctions. and the pyrone strands exchanging across the junction. The two related DNA sequences d(CCGGGACCGG) From these six crystal structures, five main principles [29••] and d(CCGGTACCGG) [30••] were designed to elu- have emerged concerning the structure and stability of cidate the effects on B-DNA of tandem G•A mismatched four-way junctions. base pairs and of cross-linking by the photochemothera- putic drug psoralen, respectively; however, both crystallized Junctions do not distort the conformation of the as near identical antiparallel Rh-X junctions. Together, stacked arms these two structures show how a fully DNA type junction is Except for the gaps in the phosphoribose backbones across stabilized by a core ACC trinucleotide sequence [30••]. the junction, the stacked duplex arms are effectively 304 Nucleic acids Figure 2 (a) RNA–DNA junctions (b) ACC trinucleotide junctions (c) Drug cross-linked junctions 5' 3' GGACAGAUGGGAG D C1 G10 5' 3' C2 G9 CCTGTCGACCCTCG C1 G10 G3 C8 Furan-side C G G C G C A A' 2 9 4 7 HMT thymine A A B' G3 C8 N5 A6 G O O R C G C7 A6 N5 C 1 8 C T 4 O O R O O T C G O ν O A G A N5 A6 C7 4 5' h G A A N C G C T 6 5 8 3 3 4' C C 4 5 O O G C7 G4 G9 2 C OH OH A B C8 G3 G10 1 Pyrone-side G C 3' 5' 9 2 thymine G10 C1 B 3' 5' d(CCGGGACCGG) A A CCAT G A C G A G CT A T F G C P P G C D C A T G C F A T G C A T G C F A G U A A P G C B F P G C G C U A G C C G G C A T d(CCGGTACCGG) d(CCGGT∗ACCGG) d(CCGCT∗AGCGG) G C Current Opinion in Structural Biology Single-crystal structures of nucleic acid junctions. (a) The RNA–DNA strands (blue and green) and their symmetry mates. The junctions [28••,31••]. The RNA chains (red) assemble with DNA to d(CCGGTACCGG) junction is formed by four strands with unique form junctions with Watson–Crick base-paired duplex arms (blue). conformations in the crystal. (c) The psoralen (drug) cross-linked One arm in each structure has only a single Watson–Crick base pair junctions [32••].
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