A global view of meiotic
double-strand break end resection
Eleni P. Mimitou,1 Shintaro Yamada,1 Scott Keeney1,2*
DNA double-strand breaks that initiate meiotic recombination are exonucleolytically
processed. This 5′→ 3′ resection is a central, conserved feature of recombination but
remains poorly understood. To address this lack, we mapped resection endpoints genome-wide at high resolution in Saccharomyces cerevisiae. Full-length resection requires Exo1
exonuclease and the DSB-responsive kinase Tel1, but not Sgs1 helicase. Tel1 also promotes
efficient and timely resection initiation. Resection endpoints display pronounced
heterogeneity between genomic loci that reflects a tendency for nucleosomes to block
Exo1, yet Exo1 also appears to digest chromatin with high processivity and at rates similar
to naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a
defining feature of the meiotic resection landscape.
Meiotic recombination promotes proper segregation of homologous chromosomes (1) and initiates with double-strand breaks (DSBs) formed by the topoisomerase-like Spo11, which remains covalently attached
to DSB 5′ ends (Fig. 1A). Endonucleolytic cleavage
by the Mre11-Rad50-Xrs2 (MRX) complex plus
Sae2 generates nicks on Spo11-bound strands
that serve as entry points for modest 3′→ 5′
Mre11 exonuclease activity and robust 5′→ 3′ Exo1
exonuclease activity (2– 5). These release Spo11
bound to short oligonucleotides (oligos) and generate 3′ single-stranded DNA (ssDNA) tails, which
are substrates for strand-exchange proteins (Dmc1
and/or Rad51) that search for homology and
invade a homologous repair template (1). A similar nick-plus-exonuclease mechanism operates
in vegetative cells ( 6), except that Exo1 is partially redundant with Sgs1-Top3-Rmi1 plus Dna2
for extensive 5′→ 3′ resection ( 7, 8). In meiosis,
available data implicate Exo1 but suggest dispensability of Sgs1 ( 4, 9).
Although meiotic resection was first demon-
strated experimentally ≥ 25 years ago and has
long been known to be a fundamental step in
recombination ( 10, 11), it remains poorly under-
stood. In fact, detailed data are available only
for one side of a single artificial DSB hotspot
( 4); whether this extrapolates to natural hotspots
is unknown. We mapped resection endpoints
genome-wide with high specificity, sensitivity,
and spatial resolution. These maps answered
long-standing questions about genetic control of
resection, the handoff from MRX-Sae2 to Exo1,
locus-to-locus variation in resection, resection
kinetics, and interaction of resection machinery
Mapping DSB resection endpoints
We digested DSB 3′ tails with ssDNA-specific
nucleases to generate sequencing libraries of
ssDNA–dsDNA junctions that we compared with
DSB maps from Spo11-oligo sequencing (Fig. 1, A
and B, and table S1) ( 12). Two biological replicates were pooled for each strain or time point
[Pearson’s correlation coefficient (r) = 0.95 to
0.99] (fig. S1, A and B).
Reads from resection endpoints should be
meiosis-specific and Spo11-dependent and should
flank DSB hotspots with defined polarity (fig.
S1C). S1 sequencing (S1-seq) reads were hotspot-enriched with the expected polarity, and enrichment was absent from premeiotic samples and
meiotic samples from the catalytically inactive
spo11-Y135F mutant (Fig. 1, B and C, and fig. S1,
D to F). S1-seq signal correlated well with Spo11
oligos (Fig. 1D), and reads spread further from
hotspots over time in dmc1D (Fig. 1B and fig. S1,
E, G, and H), reflecting known hyperresection
( 13). Thus, S1-seq is a sensitive and quantitative
measure of DSB resection endpoints.
Resection endpoints extended ~200 to 2000
nucleotides (nt) from hotspot centers (mean
822 nt), with a positive skew (Fig. 1, C and E).
This pattern resembles Southern blotting at
HIS4LEU2 (range 350–1550, mean 800 nt) (Fig.
1E) ( 4), but S1-seq captured less abundant species
at the distribution extremes. Genome-average
profiles were smooth and left-right symmetric
(Fig. 1C), but individual hotspots were heterogeneous, with peaks and valleys differing between hotspots or sides of the same hotspot
(Fig. 1B and fig. S1E).
S1-seq profiles affirmed Sgs1 dispensability
(Fig. 1, B and F, and fig. S1E). In contrast, the
nuclease-defective mutation exo1-D173A (exo1-nd)
( 14) reduced resection lengths to <1100 nt (mean
373 nt), comparable with HIS4LEU2 in exo1D
(mean 270 nt) (Fig. 1, B, E, and F, and fig. S1E)
( 4). Resection endpoints in exo1-nd likely are the
most distal Mre11-dependent nicks formed in
wild type. If so, the difference between Spo11-
oligo lengths and exo1-nd endpoints indicates
that multiple nicks are formed or that 3′→ 5′
digestion averaging 335 nt accompanies a single
distant nick (fig. S1I).
Tel1 promotes resection initiation
Absence of Tel1 [orthologous to human ataxia
telangiectasia mutated (ATM)] decreased resection length at HIS4LEU2 for early DSBs ( 15).
This and other findings led to the proposal that
Tel1 controls resection when DSB numbers are
still low, whereas higher DSB numbers later
allow Mec1 [ataxia telangiectasia and Rad3–
related (ATR) in humans] to substitute ( 15).
S1-seq data allowed us to test this model and
uncovered that Tel1 acts at multiple steps.
S1-seq reads in tel1D fell closer to DSB hotspots at early (2 hours) and later ( 4 hours) time
points, indicating shorter resection, but with
peaks similar to those of wild type (Fig. 2, A and
B). Although some tracts at 4 hours in tel1D
matched the longest in wild type (Fig. 2B), DSBs
remained hyporesected overall (Fig. 2, A and
B). Therefore, Tel1 modulates resection length
throughout meiosis, not just early.
DSBs in wild type have appeared to be maximally resected as soon as they are detectable
( 13, 15). S1-seq profiles at individual hotspots
seemed stable over time in wild type (Fig. 2A),
especially compared with dmc1D (Fig. 1B), but
genome-wide averages revealed slightly shorter
resection tracts at 2 hours than at 4 to 6 hours
(Fig. 2, B to D, and figs. S1J and S2E). Locus-to-locus variability and greater sensitivity of S1-
seq are nonexclusive possibilities for why this
change was previously unseen. Shorter tracts
may reflect partially resected DSBs, but we favor
that later-forming DSBs tend toward longer resection, perhaps via increasing Tel1 activity ( 15).
The tel1D mutant showed higher S1-seq signal
within hotspots, accounting for a greater fraction of reads at 2 hours than 4 hours in tel1D,
but also present in wild type at lower levels (Fig.
2, A and B, and fig. S1E). We hypothesized that
this signal reflects unresected DSBs. Indeed,
within-hotspot S1-seq signal displayed peaks
overlapping strong Spo11-oligo clusters (Fig. 2A)
and correlated with hotspot strength in tel1D and
wild type (fig. S2, A and B). Fine-scale patterns
matched expectation for preferred Spo11 cleavage
3′ of C residues and for the 2-nt 5′ overhang of
Spo11 primary cleavage products (fig. S2, C and
D). We conclude that unresected DSBs are present in wild type at levels difficult to detect with
Southern blotting; higher levels in tel1D indicate
that Tel1 promotes normal resection initiation,
possibly via Sae2 phosphorylation ( 16).
Accompanying resection signal, a weaker S1-seq
signal with the “wrong” polarity was seen—for
40 6 JANUARY 2017 • VOL 355 ISSUE 6320 sciencemag.org SCIENCE
1Molecular Biology Program, Memorial Sloan Kettering
Cancer Center, New York, NY 10065, USA. 2Howard Hughes
Medical Institute, Memorial Sloan Kettering Cancer Center,
New York, NY 10065, USA.
*Corresponding author. Email: firstname.lastname@example.org