Detection of Homologous Recombination Intermediates <em>via</em> Proximity Ligation and Quantitative PCR in <em>Saccharomyces cerevisiae</em>

Abstract

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, the two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway. INTRODUCTION:Homologous recombination (HR) is a high-fidelity mechanism of repair of DNA double-stranded breaks (DSBs), inter-strand cross-links, and ssDNA gaps, as well as a pathway for DNA damage tolerance. HR differs from error-prone pathways for DNA damage repair/tolerance, such as nonhomologous end-joining (NHEJ) and translesion synthesis, in that it utilizes an intact, homologous duplex DNA as a donor to template the repair event. Moreover, many of the key intermediates in the HR pathway are reversible, allowing for exquisite regulation of the individual pathway steps. During the S, G2, and M phases of the cell cycle, HR competes with NHEJ for the repair of the two-ended DSBs 1 . In addition, HR is essential to DNA replication for the repair of replicationassociated DNA damage, including ssDNA gaps and one-sided DSBs, and as a mechanism of DNA lesion bypass 2 . A critical intermediate in the HR pathway is the displacement loop, or D-loop (Figure 1). Following end resection, the central recombinase in the reaction, Rad51, loads onto the newly resected ssDNA of the broken molecule, forming a helical filament 2 . Rad51 then carries out a homology search to identify a suitable homologous donor, typically the sister chromatid in somatic cells. The D-loop is formed when the Rad51-ssDNA filament invades a homologous duplex DNA, which leads to the Watson-Crick base pairing of the broken strand with the complementary strand of the donor, displacing the opposite donor strand. Extension of the 3' end of the broken strand by a DNA polymerase repl...

Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability

“…The D-loop-Capture (DLC) (Piazza et al 2017, 2019, 2021b; Reitz et al 2022) and CR-C assays enabled the determination of the temporal succession of D-loops, MIs, and MIR1 product formation following DSB induction ( Figs. 5E-G ).…”

“…5E-G ). Improvements to the DLC protocol further enabled estimating the absolute D-loop and MI levels per haploid genome equivalent ( Methods ) (Reitz et al 2022). D-loops at the intra-chromosomal S2 donor were highest at the earliest time point assayed (2 hours) and decreased monotonically 2- to 3-fold every 2 hours ( Fig.…”

“…The DLC and DLE assays have been described previously (Piazza et al 2019, 2018) (for a step-by-step protocol, see (Reitz et al 2022)). A psoralen crosslink reversal step adapted from (Yeung et al 1988) has been added prior to the qPCR step, which removes various amplification biases and enables quantitative comparison of the amount of distinct JMs (Reitz et al 2022).…”

“…The DLC and DLE assays have been described previously (Piazza et al 2019, 2018) (for a step-by-step protocol, see (Reitz et al 2022)). A psoralen crosslink reversal step adapted from (Yeung et al 1988) has been added prior to the qPCR step, which removes various amplification biases and enables quantitative comparison of the amount of distinct JMs (Reitz et al 2022). Briefly, psoralen crosslink reversal is performed upon incubation of purified DNA in decrosslinking solution (100 mM KOH, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) at 90° C for 30 minutes.…”

“…and enables quantitative comparison of the amount of distinct JMs (Reitz et al 2022). Briefly, psoralen crosslink reversal is performed upon incubation of purified DNA in decrosslinking solution (100 mM KOH, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) at 90° C for 30 minutes.…”