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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.
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.
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....
1. Pre-growth, DSB induction, and sample collection
NOTE: Supplementation of all media with 0.01% adenine is recommended for Ade- strains.
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.......
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.......
The work in the Heyer laboratory is supported by grants GM58015 and GM137751 to W.-D.H. Research in the Piazza laboratory is supported by the European Research Council (ERC-StG 3D-loop, grand agreement 851006). D.R. is supported by T32CA108459 and the A.P. Giannini Foundation. We thank Shih-Hsun Hung (Heyer Lab) for sharing his DLC/DLE assay results and for additionally validating the changes to the assays that are detailed in this protocol.
....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 |
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