Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Summary

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

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, 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 non-homologous 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 DS....

Protocol

1. Pre-growth, DSB induction, and sample collection

NOTE: Supplementation of all media with 0.01% adenine is recommended for Ade- strains.

1. Streak out the appropriate haploid strains (see Table 1) on yeast peptone dextrose adenine (YPDA) (1% yeast extract, 2% peptone, 2% glucose, 2% agar, 0.001% adenine) and grow for 2 days at 30 °C.
2. Use a single colony to inoculate 5 mL of YPDA in a 15 mL glass culture tube. Grow cultures to saturation at 30 °C with shaking or rotation for aeration.
3. DLC assay: Prepare the 5x psoralen stock solution (0.5 mg/mL trioxsalen in 200-proof....

Results

DLC assay
The DLC assay detects both nascent and extended D-loops formed by the invasion of a site-specific DSB into a single donor (Figure 2). Psoralen crosslinking physically links the broken strand and the donor via the heteroduplex DNA within the D-loop. Restriction enzyme site restoration with a long, hybridizing oligo on the resected strand of the break allows for restriction enzyme cleavage, followed by ligation of the broken strand to the proximal dono.......

Discussion

The assays presented allow for the detection of nascent and extended D-loops (DLC assay), D-loop extension (DLE assay), and BIR product formation (DLE assay with no hybridizing oligonucleotides) using proximity ligation and qPCR. ChIP-qPCR of Rad51 to sites distant from the DSB has previously been used as a proxy for Rad51-mediated homology search and D-loop formation. However, this ChIP-qPCR signal is independent of the sequence homology between the break site and a potential donor, as well as the Rad51-associated facto.......

Materials

Name	Company	Catalog Number	Comments
1. Pre-growth, DSB induction, and sample collection
Equipment
15 and 50 mL conical tubes
15 mL glass culture tubes
250 mL, 500 mL, or 1 L flasks
60 mm x 15 mm optically clear petri dishes with flat bottom			Suggested: Corning, Catalog Number 430166; or Genesee Scientific, Catalog Number 32-150G
Benchtop centrifuge with 15 and 50 mL conical tube adapters
Benchtop orbital shaker or tube rotator/revolver
Rotator
UV crosslinker or light box with 365 nm UV bulbs set atop an orbital shaker			Suggested: Spectrolinker XL-1500 UV Crosslinker (Spectronics Corporation) or Vilbert Lourmat BLX-365 BIO-LINK, Catalog Number 611110831
Materials
60% w/w sodium DL-lactate syrup	Sigma-Aldrich	L1375	For media preparation
Agar	Fisher	BP1423500	For media preparation
Bacto peptone	BD Difco	211840	For media preparation
Bacto yeast extract	BD Difco	212750	For media preparation
D-(+)-glucose	BD Difco	0155-17-4	For media preparation
Trioxsalen	Sigma-Aldrich	T6137	For psoralen cross-linking
2. Spheroplasting, lysis, and restriction enzyme site restoration
Supplies
1.5 mL microcentrifuge tubes
Dry bath
Liquid nitrogen or dry ice/ethanol
Refrigerated microcentrifuge or microcentrifuge
Materials
10X restriction enzyme (CutSmart) buffer (500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 1 mg/mL BSA, pH 7.8-8.0)
Zymolyase 100T	US Biological	Z1004	For spheroplasting
3. Restriction enzyme digest and intramolecular ligation
Supplies
Water bath
Materials
EcoRI-HF	New England Biolabs	R3101	Restriction enzyme digest for DLC assay
HindIII-HF	New England Biolabs	R3104	Restriction enzyme digest for DLE assay
T4 DNA ligase	New England Biolabs	M0202	Intramolecular ligation
4. DNA purification
Supplies
1.5 and 2 mL microcentrifuge tubes
Materials
Phenol/chloroform/isoamyl alcohol (P/C/IA) at 25:24:1	Sigma-Aldrich	P2069	DNA purification
5. Psoralen cross-link reversal
Supplies
Thermocycler/PCR machine
6. qPCR
Supplies
Lightcycler 480	Roche	5015278001	qPCR machine used by the authors
Lightcycler 96	Roche	5815916001	qPCR machine used by the authors
Materials
LightCycler 480 96-Well Plate, white	Roche	4729692001	96-well plates for qPCR
SsoAdvanced Universal SYBR Green Super Mix	BioRad	1725271	qPCR kit used by the authors
SYBR Green I Master Mix	Roche	4707516001	qPCR kit used by the authors