To detect meiotic DSBs in S. cerevisiae, genomic DNA is extracted from synchronously sporulating cultures, then digested with restriction enzyme(s) as necessary to yield an appropriate sized fragment. Subsequently, these DNA fragments are separated by gel electrophoresis, and both the parental length (unbroken) DNA and the DSB fragments are detected by Southern blotting and indirect end-labeling by hybridization to an appropriate probe ().
The procedure of DSB mapping at four different resolutions.
Meiotic DSBs do not occur randomly throughout the genome. Instead, DSBs (and the resulting recombination products) form preferentially within small (≤2 kbp) regions called hotspots (1
). DSBs show specificfeaturesof distribution according to the resolution of the methods used to detect them. When observed at the resolution of a whole chromosome (100’s to 1000’s of kbp in S. cerevisiae
), alternating hot and cold domains are observed that are typically on the order of 50–100 kbp wide (see
for an example). When examined at the resolution of individual genes (i.e., examining ~5–20 kbp at a time), individual hotspots can be observed that are separated by several kbp of DNA in which few, if any, DSBs are formed (). Most hotspots in S. cerevisiae
are located within transcriptional promoter regions (2
). Mapping at yet higher resolution reveals that each hotspot consists of multiple DSB sites clustered within regions of ~75–250 bp (). Finally, mapping at the resolution of individual nucleotides reveals that Spo11 cleaves the DNA to yield a two-nucleotide 5′ overhang and that some positions within hotspots are cut more frequently than others, although no obvious DNA sequence preference has yet emerged (3
Chromosome level mapping.
Medium and high resolution mapping of DSB in a rad50S strain.
Because there is no single method for separating and detecting DNA fragments across all of the size ranges outlined above, it is necessary to choose DSB mapping strategies appropriate to the purpose of the experimental study. Here we describe procedures for DSB analysis at four levels of spatial resolution, which we define as chromosome level, medium resolution, high resolution, and nucleotide level mapping. These differ from one another primarily with respect to DNA sample preparation and the method of gel electrophoresis (pulsed-field, conventional agarose, polyacrylamide, or sequencing gel, respectively). These protocols are modified from established methods (5
In many studies, the position and the frequency of meiotic DSBs are measured using rad50S
mutants, which are deficient in a process of endonucleolytic release of covalently attached Spo11 from DSB ends (5
). These mutants accumulate unrepaired DSBs, making observation of the normally transient meiotic DSB much easier. The frequency of DSBs varies from one hotspot to another and, in general, DSB frequencies measured in rad50S
strains correlate well with overall recombination frequencies in corresponding RAD50+
. However, it is important to note that DSB frequencies are substantially reduced in rad50S
mutants in regions where replication is experimentally delayed (8
), and recent genome-wide studies demonstrate that DSBs in certain regions are specifically underreported in rad50S
-like mutants (11
To obtain well-synchronized meiotic samples, cells are precultured in presporulation medium (SPS), and then transferred to sporulation medium (SPM). In the SK1 strain background, the DSB frequency reaches maximum levels about 5–6 h after transfer to SPM. Premeiotic samples are harvested at 0 h (i.e., immediately upon transfer to SPM), and meiotic samples are harvested at appropriate time points thereafter (seeNote 1
The frequencies of meiotic DSBs are low, so it is important that genomic DNA is extracted gently to avoid mechanical shearing. Excessive shearing generates a high background of hybridizing signal in lanes from the gel electrophoresis, and can obscure weaker DSB signals and/or make DSBs difficult to quantify. DNA samples in solution should be mixed gently, and vigorous pipetting and vortexing should be avoided.
To observe DSBs at different levels of spatial resolution, the lengths of the parental and DSB fragments need to be considered, and an appropriate probe needs to be chosen for indirect end-labeling of Southern blots. Parental and DSB fragments should be well separated by gel electrophoresis in order to accurately estimate DSB frequencies. It is recommended that probes are designed within open reading frames so that the region to which the probe hybridizes is unlikely to itself be a site of DSB formation. Additional specifications are discussed in the individual methods sections below.