The Holliday junction in an inverted repeat DNA sequence: Sequence effects on the structure of four-way junctions Brandt F. Eichman, Jeffrey M. Vargason, Blaine H. M. Mooers, and P. Shing Ho* Department of Biochemistry and Biophysics, ALS 2011, Oregon State University, Corvallis, OR 97331-7305 Communicated by K. E. van Holde, Oregon State University, Corvallis, OR, December 9, 1999 (received for review November 8, 1999) Holliday junctions are important structural intermediates in recom- bination, viral integration, and DNA repair. We present here the single-crystal structure of the inverted repeat sequence d(CCGG- TACCGG) as a Holliday junction at the nominal resolution of 2.1 Å. Unlike the previous crystal structures, this DNA junction has B-DNA arms with all standard Watson–Crick base pairs; it therefore rep- The Hollidayresents the intermediate junction proposed by Holliday as beingin involved an in inverted repeat DNA homologous recombination. The junction is in the stacked-X con- formation, with two interconnected duplexes formed by coaxially stacked arms, and is crossed at an angle of 41.4° as a right-handed sequence:X. A Sequence sequence comparison with previous effects B-DNA and junction on the structure of crystal structures shows that an ACC trinucleotide forms the core of a stable junction in this system. The 3؅-C⅐G base pair of this ACC core forms direct and water-mediated hydrogen bonds to the four-wayphosphates junctions at the crossover strands. Interactions within this core define the conformation of the Holliday junction, including the angle relating the stacked duplexes and how the base pairs are Brandt F. Eichman, Jeffreystacked in M.the stable Vargason, form of the junction. Blaine H. M. Mooers, and P. Shing Ho* Department of BiochemistryDNA and structure Biophysics,͉ recombination ALS 2011, Oregon State University, Corvallis, OR 97331-7305 Fig. 1. Conformations of four-way junctions. (Left) Association of DNA strands A (blue), B (green), C (red), and D (yellow) to form a junction (Upper) hen genetic information is exchanged, e.g., during recom- Communicated by K. E. van Holde, Oregon State University, Corvallis, OR, Decemberwith four 9, duplex 1999 arms (received extended in afor square review planar geometry November (extended-X 8, 1999) Wbination between homologous regions of chromosomes or form, Lower). (Right) These same strands (Upper) associated to form the integration of viral DNA into host genomes, the DNA double stacked-X structure of the junction, with pairs of arms coaxially stacked as Holliday junctions are importanthelix is disrupted. structural Holliday intermediates proposed that the in intermediate recom- double helices related by 2-fold symmetry (Lower). formed during homologous recombination is a four-way junction bination, viral integration,(Fig. 1) and (1). Similar DNA junctions repair. form We in cruciform present DNAs here extruded the single-crystal structurefrom of the inverted inverted repeat sequences. repeat Recently, sequence the crystal d(CCGG- structures Despite repeated efforts over the years to crystallize a four-way of junctions in a DNA–RNA complex (2) and in the sequence DNA junction, the recent crystal structures have all been seren- TACCGG) as a Holliday junctiond(CCGGGACCGG) at the (3) nominal have been resolution reported. In the of first 2.1 struc- Å. dipitous. The RNA͞DNA junction resulting from studies on an BIOPHYSICS ture, the DNA͞RNA arms are in the A-conformation, whereas RNA-cleaving DNA motif, or DNAzyme, complexed with its RNA Unlike the previous crystalin the structures, latter, two G⅐Amismatchedbasepairssitadjacenttothe this DNA junction has B-DNA substrate (2) have arms that adopt an A-RNA conformation. The sequence d(CCGGGACCGG) designed to study tandem G⅐A crossover between the duplexes. Here, we present the structure arms with all standard Watson–Crick base pairs; it therefore rep- mismatched base pairs in B-DNA also crystallized as a junction (3); of a Holliday junction in a true inverted repeat DNA sequence however, the structure around the junction is perturbed by the resents the intermediated(CCGGTACCGG) proposed by in Holliday which all of as the being nucleotides involved are in B-type in mismatches. Thus we are left asking what is the structure of a homologous recombination.helices with The Watson–Crick junction base is in pairs. the stacked-X con- Holliday junction with B-DNA arms and standard base pairs. Studies on synthetic four-stranded complexes and DNA cru- We had designed the sequence d(CCGGTACCGG) to study formation, with two interconnectedciforms show that four-way duplexes junctionsDNA formed can Four- adopt by eitherways coaxially an openjunctionsthe d(TA) dinucleotide in B-DNA that is a target for the extended-X or the more compact stacked-X conformations (for photochemotherapeutic drug psoralen. Surprisingly, four stacked arms, and is crossedarecentreview,seeref.4).Inthepresenceofmonovalent at an angle of 41.4°« Holliday as a right-handed junctions » strands of this DNA assembled to crystallize as a four-way X. A sequence comparisoncations, the with four arms previous of the junction B-DNA are extended and into junction a square junction with all Watson–Crick base pairs. We can thus examine planar geometry (Fig. 1) to minimize electrostatic repulsion the detailed structure and define the nucleotides and intramo- crystal structures showsbetween that phosphates. an ACC Divalent trinucleotide cations and polyvalent forms the polyamines core lecular interactions that help to stabilize the Holliday junction. help shield the phosphate charges (5), allowing the junction to of a stable junction in thisadopt system. a more compact The 3 structure؅-C⅐G base with pairs pair of of arms this coaxially ACC Materials and Methods core forms direct andstacked water-mediated as duplexes (Fig. 1) hydrogen and the duplexes bonds related to by ϳ the60° Crystallization and X-Ray Data Collection. DNA sequences were (6–9). A 63° angle is estimated from atomic force microscopy synthesized on an Applied Biosystems DNA synthesizer and phosphates at the crossoverstudies on strands. arrays of such Interactions junctions (10). withinDuring recombination, this core four-way junctions are resolved by enzymes to complete the define the conformation of the Holliday junction, including the Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, process of strand exchange between duplexes. The junctions seen www.rcsb.org {PDB ID codes 1DCW [d(CCGGTACCGG)] and 1DCV [d(CCGCTAGCGG)]}. angle relating the stackedin cocrystals duplexes with the and resolving how enzymes the RuvA base (11, pairs 12) and are Cre *To whom reprint requests should be addressed. E-mail: [email protected]. (13) are in the extended-X form, whereas T4 endonuclease VII stacked in the stable form of the junction. The publication costs of this article were defrayed in part by page charge payment. This (14, 15) and T7 endonuclease I (16, 17) seem to maintain the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. relationship of the stacked-X arms. §1734 solely to indicate this fact. DNA structure ͉ recombination PNAS ͉ April 11, 2000 ͉ vol. 97 ͉ no. 8 ͉ 3971–3976 Fig. 1. Conformations of four-way junctions. (Left) Association of DNA • These structures have been described in 1964 by Robin Holliday strands A (blue), B (green), C (red), and D (yellow) to form a junction (Upper) hen genetic information is exchanged, e.g.,• Key during intermediatestructure recom- in many types of genetic recombination, as well as in double-strand break repairwith four duplex arms extended in a square planar geometry (extended-X Wbination between homologous regions of• chromosomes Natural 4-ways junctions or haveform, a symmetricalLower). (Right sequence) These and same are thus strands mobile, ( Upper) associated to form the integration of viral DNA into host genomes, themeaning DNA that double the four individualstacked-X arms structure may slide through of the the junction, junction with pairs of arms coaxially stacked as helix is disrupted. Holliday proposed that the intermediate double helices related by 2-fold symmetry (Lower). formed during homologous recombination is a four-way junctionNANOANDES 2017, November 22-29, Buenos Aires, Argentina 41 (Fig. 1) (1). Similar junctions form in cruciform DNAs extruded from inverted repeat sequences. Recently, the crystal structures Despite repeated efforts over the years to crystallize a four-way of junctions in a DNA–RNA complex (2) and in the sequence DNA junction, the recent crystal structures have all been seren- d(CCGGGACCGG) (3) have been reported. In the first struc- dipitous. The RNA͞DNA junction resulting from studies on an BIOPHYSICS ture, the DNA͞RNA arms are in the A-conformation, whereas RNA-cleaving DNA motif, or DNAzyme, complexed with its RNA in the latter, two G⅐Amismatchedbasepairssitadjacenttothe substrate (2) have arms that adopt an A-RNA conformation. The sequence d(CCGGGACCGG) designed to study tandem G⅐A crossover between the duplexes. Here, we present the structure mismatched base pairs in B-DNA also crystallized as a junction (3); of a Holliday junction in a true inverted repeat DNA sequence however, the structure around the junction is perturbed by the d(CCGGTACCGG) in which all of the nucleotides are in B-type mismatches. Thus we are left asking what is the structure of a helices with Watson–Crick base pairs. Holliday junction with B-DNA arms and standard base pairs. Studies on synthetic four-stranded complexes and DNA cru- We had designed the sequence d(CCGGTACCGG) to study ciforms show that four-way junctions can adopt either an open the d(TA) dinucleotide in B-DNA that is a target for the extended-X or the more compact stacked-X conformations (for photochemotherapeutic drug psoralen. Surprisingly, four arecentreview,seeref.4).Inthepresenceofmonovalent strands of this DNA assembled to crystallize as a four-way cations, the four arms of the junction are extended into a square junction with all Watson–Crick base pairs.