Abstract
Displacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loops with near base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.
Highlights
Homologous recombination (HR) is a universal DNA repair pathway that is vital to genome maintenance and the repair of double-stranded DNA breaks, stalled or collapsed forks and inter-strand crosslinks (Kowalczykowski et al, 2016; San Filippo et al, 2008; Wright et al, 2018)
For every 10.4 bp of heteroduplex DNA (hDNA) generated in the Displacement loops (D-loops), one negative supercoil from the dsDNA is consumed
Plasmids purified from E. coli have a mean supercoiling density of s = À0.07 supercoil/turn (Champion and Higgins, 2007), which we verified independently (Solinger et al, 2002). Three kbp plasmids such as those used in this study should contain ~20 ± 5 negative supercoils, meaning that they can accommodate a maximum of ~210 ± 50 bp hDNA, even if the length of the invading homologous single-stranded DNA (ssDNA) is longer
Summary
Homologous recombination (HR) is a universal DNA repair pathway that is vital to genome maintenance and the repair of double-stranded DNA breaks, stalled or collapsed forks and inter-strand crosslinks (Kowalczykowski et al, 2016; San Filippo et al, 2008; Wright et al, 2018). The Rad protein forms a filament on the ssDNA and carries out DNA strand invasion into a homologous duplex donor. The Rad motor protein translocates along the three-strand intermediate that has been formed by the invasion (Wright and Heyer, 2014). It simultaneously displaces Rad, while threading out a heteroduplex DNA (hDNA) and a displaced strand. The second end of the break can anneal to the displaced strand of the extended D-loop, leading to the formation of a double-Holliday junction (dHJ) (Kowalczykowski, 2015; Paques and Haber, 1999). Crossover products are disfavored in somatic cells as they have the potential to lead to genomic rearrangements or loss of heterozygosity (Li and Heyer, 2008)
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