DNA Holliday junctions are important structural intermediates in recombination, viral integration and DNA repair. We had previously determined the single-crystal structure of a four-way HoIIiday junction in an inverted repeat sequence. [Other sequences crystallize as standard B-DNA duplexes in this same crystal system] From this study, a d(ApCpC) trinucleotide was identified as the core of the stable junction in this system The significance of this sequence motif has been confirmed by more recent studies on drug-bound and methylated junctions in this laboratory.
The aim of the current proposal is to use this basic crystal system to characterize the effect of sequence and cations on the structure and stability of DNA junctions. [In addition, we propose to determine whether this sequence motif and the details of the conformation observed in the crystal system are also I relevant to Holliday junctions in solution.] The long range goal is to define the factors that stabilize and fix the four-way junction, and to understand how the junctions may serve as sites for the exchange of genetic information and for recognition by DNA repair enzymes. Studies are proposed to identify all trinucleotide sequences that crystaIIize as Holliday junctions and, therefore, provide detailed structural information on the effect of sequence on this recombination intermediate. Related to this, we will study the contribution of substituent groups in the major and minor grooves of the stacked duplex arms on the ability of these junctions to form in this crystal system. The contribution of the direct and solvent mediated hydrogen bonding interactions within the ACC-core on the formation of Holliday junctions will be studied using a crvstallographic competition assay on DNAs with phosphorothiate linkages. A set of crystallographic studies is proposed to map and characterize the specific interactions of junctions with monovalent and divalent metal ions, and the polyvalent cations spermine and spermidine. [A series of gel electrophoresis studies are proposed to relate the sequence and structural effects seen in the crystals with effects in solution. In the first set of studies, the concentration dependent formation of junctions will be examined. The assay will be used to 1) directly determine the effect of the ACC-core on the formation of junctions and 2) to screen though an in vitro evolution process alI trinucleotide sequences that stabilize junctions in solution.] In a separate set of experiments, a two-dimensional gel electrophoresis assay will be used to quantify the contributions of the molecular interactions seen in the crystals to the thermodynamic stability of four-way junctions formed at the base of cruciform DNAs extruded from negatively supercoiled plasmid DNAs. Finally, a set of studies are designed to determine whether the geometric relationship between the stacked arms across the junction is dependent on the sequence or on crystal interactions. From these studies, we will define the relevant intramolecular and solvent interactions that are responsible for fixing the location and geometry of four-way Holliday junctions.
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