Schematic of the looped dsDNA unfolding experiments for the case of (a) no pairing and (b) complete pairing. The construct is built from 50 kb λ-phage dsDNA ligated at the end to a 10 kb homologous fragment (for details see the main text). Here, the red lines represent the part of dsDNA which is not identical in sequence to the 10 kb fragment. The two 10 kb sequences which are identical to each other are shown in green to yellow shading to indicate which way the two sequences run (the yellow indicating the end sequence of the λ-phage DNA). When the constructs are fully extended the two homologous pieces run in opposite directions, but when folded any paired tracts will run in the same direction. Shown in (b) is a coil–expected form for the DNA – where the magnification shows that, where pairing occurs, two sections may align so that their identical sequences read in the same direction. (c) Is a representative result showing the extension versus force curves (decreasing force only) in 150 mM NaCl–PBS at 25°C for a dsDNA construct (solid) and a control curve for a 60 kb dsDNA in the same ionic conditions (dashed). The y-axis is normalized to L19, the measured extension of the controls at 19 pN. Further experimental data curves for different ionic conditions are given in the electronic supplementary material, S1. (d) The solid line shows the difference between the two curves (here, defined as the extension of the construct minus the experimentally measured control) shown in (c), multiplied by L19, resulting in negative values since the extension of the paired dsDNA is shorter than the 60 kb control. The dotted line shows the standard deviation for 25 controls. At forces less than 2 pN, the constructs containing homologous regions show significant variation, but even at 5 pN the difference between the constructs and controls is more than 10 times the standard deviation in the controls. The results indicate a significant interaction between identical DNA tracts, which affects the extension–force curve even when the applied force exceeds 10 pN. Experiments for higher temperatures (37°C and 40°C) have also been performed that are not shown here; they demonstrate qualitatively similar effect of ‘homology recognition’; the data for these temperatures are treated in the electronic supplementary material, S5. (Online version in colour.)
A new study provides more evidence that identical sections of DNA can match up with each other without the help of other molecules. Genes are regularly damaged and need to be repaired. During one repair process, called homologous recombination, a damaged, broken gene is replaced with the same gene of a spare intact DNA copy. Errors due to wapping ‘wrong’ genes lead to various genetic disorders, such as the condition progeria that causes rapid aging.
As well as repairing genes, homologous recombination is crucial in reproduction and evolution, as it allows genes that we get from each parent to be shuffled together. So-called recombination proteins do the copying and replacement of genes in this process. While proteins are responsible for the recombination process, it was not known how homologous genes find each other beforehand. There are more than 20,000 genes in human DNA coiled in up into chromosomes, so finding the homologous gene on a different DNA strand is no easy task.
Previous experiments found DNA molecules with the same genetic code are able to find their matches without outside help – no proteins etc to guide them towards each other. Those experiments showed that two sets of relatively short identical double-stranded DNA fragments, randomly mixed in a solution, would spontaneously group together with those of the same type. A later study by researchers at Harvard University found further evidence for pairing of longer homologous double-stranded DNA.
Now, in a new joint Harvard and Imperial study the team present evidence of the attraction between long identical DNA sections within a single molecule. The Harvard team anchored one end of a dsDNA to the bottom of a very small container of solution, and attached a magnetic bead to the other end. This allowed them to manipulate the molecule and pull it straight using a magnet.
They constructed dsDNA molecules with long sections carrying the same genetic code next to each other and running in opposite directions – head-to-head – and compared their behaviour to DNA without identical sections. In pulling the magnetic bead away from the bottom of the container at a fixed force, molecules with matching sections folded in on themselves and formed a loop, thereby pairing identical genes.
Dr Lee said: “Having proved an interaction between identical sequences in single DNA molecules, in these new Harvard-based experiments, the next step would be to further probe the recognition mechanism. Independently of this, we also hope that the new result will trigger experiments in real cells, to see if the recognition process occurs in the same way naturally for homologous genes.” http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_19-7-2016-15-58-56
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