Biology subjects

Scott Keeney

Publications and source records attributed to Scott Keeney.

4 recordsLinked to original sources

bioRxiv2016

A global view of meiotic double-strand break end resection

The DNA double-strand breaks that initiate homologous recombination during meiosis are subject to extensive 5'[->]3' exonucleolytic processing. This resection is a central and conserved feature of recombination, yet its mechanism is poorly understood. Using a purpose-made deep-sequencing method, we mapped meiotic resection endpoints genome-wide at high spatial resolution in Saccharomyces cerevisiae. Generating full-length resection tracts requires Exo1 exonuclease activity and the DNA-damage responsive kinase Tel1, but not the helicase Sgs1. Tel1 is also required for efficient and timely initiation of resection. We find that distributions of resection endpoints at individual genomic loci display pronounced heterogeneity that reflects a tendency for nucleosomes to block Exo1 in vivo, yet modeling experiments indicate that Exo1 digests chromatin with high apparent processivity and at rates approaching those for naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a determining feature of the meiotic resection landscape.

Molecular Biology↗

bioRxiv2016

Numerical and Spatial Patterning of Yeast Meiotic DNA Breaks by Tel1

The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and distribution. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to examine the contribution of the DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM) to this regulation in Saccharomyces cerevisiae. A tel1{Delta} mutant had globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tell mutation also increased Spo11-oligonucleotide levels, but mutating known Tel1 phosphotargets on Hop1 and Rec114 did not. Deep sequencing of Spo11 oligonucleotides from tel1{Delta} mutants demonstrated that Tel1 shapes the nonrandom DSB distribution in ways that are distinct but partially overlapping with previously described contributions of the recombination regulator Zip3. Finally, we uncover a context-dependent role for Tel1 in hotspot competition, in which an artificial DSB hotspot inhibits nearby hotspots. Evidence for Tel1-dependent competition involving strong natural hotspots is also provided.

Molecular Biology↗

bioRxiv2015

Non-paradoxical evolutionary stability of the recombination initiation landscape in Saccharomycetes

The nonrandom distribution of meiotic recombination shapes heredity and genetic diversification. A widely held view is that individual hotspots -- favored sites of recombination initiation -- are always ephemeral because they evolve rapidly toward extinction. An alternative view, often ignored or dismissed as implausible, predicts conservation of the positions of hotspots if they are chromosomal features under selective constraint, such as gene promoters. Here we empirically test opposite predictions of these theories by comparing genome-wide maps of meiotic recombination initiation from widely divergent species in the Saccharomyces clade. We find that the frequent overlap of hotspots with promoters is true of the species tested and, consequently, hotspot positions are well conserved. Remarkably, however, the relative strength of individual hotspots is also highly conserved, as are larger-scale features of the distribution of recombination initiation. This stability, not predicted by prior models, suggests that the particular shape of the yeast recombination landscape is adaptive, and helps in understanding evolutionary dynamics of recombination in other species.

Evolutionary Biology↗

bioRxiv2015

Local and sex-specific biases in crossover vs. noncrossover outcomes at meiotic recombination hotspots in mouse

Meiotic recombination initiated by programmed double-strand breaks (DSBs) yields two types of interhomolog recombination products, crossovers and noncrossovers, but what determines whether a DSB will yield a crossover or noncrossover is not understood. In this study we analyze the influence of sex and chromosomal location on mammalian recombination outcomes by constructing fine-scale recombination maps in both males and females at two mouse hotspots located in different regions of the same chromosome. These include the most comprehensive maps of recombination hotspots in oocytes to date. One hotspot, located centrally on chromosome 1, behaved similarly in male and female meiosis: crossovers and noncrossovers formed at comparable levels and ratios in both sexes. In contrast, at a distal hotspot crossovers were recovered only in males even though noncrossovers were obtained at similar frequencies in both sexes. These findings reveal an example of extreme sex-specific bias in recombination outcome. We further find that estimates of relative DSB levels are surprisingly poor predictors of relative crossover frequencies between hotspots in males. Our results demonstrate that the outcome of mammalian meiotic recombination can be biased, that this bias can vary depending on location and cellular context, and that DSB frequency is not the only determinant of crossover frequency.

Genetics↗