Single molecule detection of direct, homologous, DNA/DNA pairing

Abstract

Using a parallel single molecule magnetic tweezers assay we demonstrate homologous pairing of two double-stranded (ds) DNA molecules in the absence of proteins, divalent metal ions, crowding agents, or free DNA ends. Pairing is accurate and rapid under physiological conditions of temperature and monovalent salt, even at DNA molecule concentrations orders of magnitude below those found in vivo, and in the presence of a large excess of nonspecific competitor DNA. Crowding agents further increase the reaction rate. Pairing is readily detected between regions of homology of 5 kb or more. Detected pairs are stable against thermal forces and shear forces up to 10 pN. These results strongly suggest that direct recognition of homology between chemically intact B-DNA molecules should be possible in vivo. The robustness of the observed signal raises the possibility that pairing might even be the ''default'' option, limited to desired situations by specific features. Protein-independent homologous pairing of intact dsDNA has been predicted theoretically, but further studies are needed to determine whether existing theories fit sequence length, temperature, and salt dependencies described here.dsDNA ͉ sequence-dependent P airing of homologous DNA/chromosome regions is a central feature of many biologically important processes. Recombinational double-strand break repair and programmed homologous recombination during meiosis all involve complex series of biochemical reactions in which single-stranded DNA (ssDNA) plays a prominent role. There also exist homologous pairing reactions that seem to involve interactions between chromosomal regions whose DNAs are chemically intact double-stranded DNA (dsDNA) (1-15). In some instances, whole chromosomes pair via multiple interactions all along their lengths (1-9) or via any region present in duplicate copies (10). In other cases, pairing occurs preferentially or exclusively in particular localized regions (''pairing sites''), which tend to involve repeated sequences, specific proteins (for establishment and/or maintenance of pairing) and/or heterochromatic regions (characterized by a paucity of genes and a less ''open'' chromatin structure) (1,(11)(12)(13)(14)(15).In contrast to recombination-related processes that are known to involve protein-mediated Watson-Crick basepairing interactions between a ssDNA and a ssDNA or dsDNA partner, the fundamental basis for ''recombination-independent'' pairing remains mysterious. The most obvious possibility is direct DNA/DNA interactions. Theoretical models have proposed that homology recognition arises from non-Watson-Crick hydrogen bond interactions between bases in the major or minor grooves (16). Local melting could also occur, permitting recognition via standard Watson-Crick base pairing. Other theories suggest that homology recognition can occur due to interactions between sequencedependent charge distributions associated with neighboring DNA helices, where the charge distributions include not only the phosphates in the DNA but also other ...

Excluded volume effect enhances the homology pairing of model chromosomes

“…It should be noted that homologous loci can often be bound by the electrostatic forces or the bindings of DNA-bridging proteins if these regions are sufficiently close, as mentioned in recent studies [1][2][3][4][5][6][7][8][9]. Thus, if homologous chromosomes are close enough for an appropriately long period of time, as observed in the present results for large W values, the synapsis of homologous loci can form with a sufficiently high probability.…”

“…Figure 5 (2,5), and (3, 6)), and < ... > samples indicates the average over 12 different simulation results using different random numbers generating η n i (t). In the case of larger K values, the pairing of homologous polymers still occurs but the process is slowed down and thus takes a much longer time than in the case with an appropriate K value.…”

“…In this case, the homologous polymers become closer together over time, and in the final stage, the system relaxes to the state in which the homologous polymers are partially stacked on top of one another. In order to characterize the temporal evolution of the distance between each pair of homologous chromosomes, we measured D nm (t) = N n i |x n i − x m i |/N n , where the n-th and m-th polymers are the homologous polymers ((n, m) = (1, 4), (2,5), and (3,6), and N m = N n ), x n i , and x m i are the positions of the corresponding particles between these polymers. As shown in Fig.…”

“…This process requires synapsis formation between homologous loci along the lengths of maternal and paternal chromosomes. Recent theoretical studies suggest that the homologies of the sequence-dependent distributions of the electrostatic charge and the binding sites of DNA-bridging proteins play important roles in the recognition and pairing of homologous loci [1][2][3][4][5][6][7][8][9].…”

Which way up? Recognition of homologous DNA segments in parallel and antiparallel alignments

“…The experiments utilized gel electrophoresis, 37 cholesteric liquid crystals, 38 AFM imaging, 39 and surface-confined DNA using magnetic beads. 40 In addition, Seeman's group reported the formation of PX-DNA complexes presumably resulting from homologue pairing, 41 and homology recognition has also been observed between nucleosomes. 39 In two recent publications, 42,43 in which the forces that depend on helix structure were considered, the effect of sliding one molecule with respect to another on interaction energies between homologous sequences, aligned in the same direction, was investigated.…”