DNA molecules can drive the assembly of other DNA molecules into specific four-stranded structures

Abstract

Single-stranded DNA molecules containing clustered G-repeats can be assembled into various four-stranded structures linked by G-quartets. Here, we report that such molecules can also drive the assembly of other DNA molecules containing G-repeats into specific four-stranded structures. In these assays, the oligonucleotides 5′-CAGGCTGAGCAGGTAC GGGGG AGCT GGGG TAGATTGGAATGTAG-3′ (oligo D) and 5′-C GGGGG AGCT GGGG T-3′ (oligo B), consisting of sequences found in immunoglobulin switch regions, were annealed in a buffer containing K + and the annealing products were analyzed by polyacrylamide gel electrophoresis. This analysis revealed that whereas annealing of each oligo alone produced four-stranded structures designated D 2 and B 2, annealing of mixtures containing both oligos produced additional complexes designated D 2∗ and B 2∗. D 2∗ and B 2∗ were found to contain only D molecules and only B molecules, respectively. The yield of D 2∗ increased and the yield of 2∗ decreased, as the concentration ratio oligo B/oligo D was increased. These results indicated that B can drive the assembly of D into D 2∗ and D can drive the assembly of B into B 2∗. Further studies revealed that while the assembly of D 2 followed a second order kinetics, the B-driven assembly of D 2∗ followed a first order kinetics. Dimethyl sulfate footprinting indicated that both D 2and D 2∗ are four-stranded structures containing two parallel and two antiparallel chains. In addition, annealing of D mixed with various B mutants showed that only mutants containing two G-clusters can drive the assembly of D 2∗. Based on these data, we propose that in the process of D 2∗ assembly, a four-stranded intermediate containing B and D is formed and then dissociates into D 2∗ and B in a rate-limiting first order reaction. Driver mechanisms of this type may cause formation of specific four-stranded structures at G-rich chromosomal sites, thereby regulating processes such as recombination and telomere synthesis.