Repair of mismatched templates during Rad51-dependent Break-Induced Replication.

Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3' end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. BIR still occurs about 10% as often when every 6th base is mismatched as with a perfectly matched donor. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, each with different spatial arrangements. Although 2 of the 6 arrangements we tested were nearly as efficient as the evenly-spaced reference, 4 were significantly less efficient. A donor with all 10 mismatches clustered at the 3' invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5' end. Our data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but mismatch position-specific effects are also important. We also addressed an apparent difference between in vitro and in vivo strand exchange assays, where in vitro studies had suggested that at a single contiguous stretch of 8 consecutive bases was needed to be paired for stable strand pairing, while in vivo assays using 108-bp substrates found significant recombination even when every 6th base was mismatched. Now, using substrates of either 90 or 108 nt-the latter being the size of the in vivo templates-we find that in vitro D-loop results are very similar to the in vivo results. However, there are still notable differences between in vivo and in vitro assays that are especially evident with unevenly-distributed mismatches. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3' end. Mismatch incorporation depends on the 3'→ 5' proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins, or from Rad1-Rad10 flap nuclease or the Mph1 helicase. Surprisingly, the probability of a mismatch 27 nt from the 3' end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase δ "chews back" the 3' end of the invading strand without any mismatch-dependent cues from the strand invasion structure. However, there appears to be an alternative way to incorporate a mismatch at the first base at the 3' end of the donor.

Yeast ATM and ATR kinases use different mechanisms to spread histone H2A phosphorylation around a DNA double-strand break.

One of the hallmarks of DNA damage is the rapid spreading of phosphorylated histone H2A (γ-H2AX) around a DNA double-strand break (DSB). In the budding yeast Saccharomyces cerevisiae, nearly all H2A isoforms can be phosphorylated, either by Mec1ATR or Tel1ATM checkpoint kinases. We induced a site-specific DSB with HO endonuclease at the MAT locus on chromosome III and monitored the formation of γ-H2AX by chromatin immunoprecipitation (ChIP)-qPCR in order to uncover the mechanisms by which Mec1ATR and Tel1ATM propagate histone modifications across chromatin. With either kinase, γ-H2AX spreads as far as ∼50 kb on both sides of the lesion within 1 h; but the kinetics and distribution of modification around the DSB are significantly different. The total accumulation of phosphorylation is reduced by about half when either of the two H2A genes is mutated to the nonphosphorylatable S129A allele. Mec1 activity is limited by the abundance of its ATRIP partner, Ddc2. Moreover, Mec1 is more efficient than Tel1 at phosphorylating chromatin in trans-at distant undamaged sites that are brought into physical proximity to the DSB. We compared experimental data to mathematical models of spreading mechanisms to determine whether the kinases search for target nucleosomes by primarily moving in three dimensions through the nucleoplasm or in one dimension along the chromatin. Bayesian model selection indicates that Mec1 primarily uses a three-dimensional diffusive mechanism, whereas Tel1 undergoes directed motion along the chromatin.

Chromosome-refolding model of mating-type switching in yeast.

Chromosomes are folded into cells in a nonrandom fashion, with particular genetic loci occupying distinct spatial regions. This observation raises the question of whether the spatial organization of a chromosome governs its functions, such as recombination or transcription. We consider this general question in the specific context of mating-type switching in budding yeast, which is a model system for homologous recombination. Mating-type switching is induced by a DNA double-strand break (DSB) at the MAT locus on chromosome III, followed by homologous recombination between the cut MAT locus and one of two donor loci (HMLα and HMRa), located on the same chromosome. Previous studies have suggested that in MATa cells after the DSB is induced chromosome III undergoes refolding, which directs the MAT locus to recombine with HMLα. Here, we propose a quantitative model of mating-type switching predicated on the assumption of DSB-induced chromosome refolding, which also takes into account the previously measured stochastic dynamics and polymer nature of yeast chromosomes. Using quantitative fluorescence microscopy, we measure changes in the distance between the donor (HMLα) and MAT loci after the DSB and find agreement with the theory. Predictions of the theory also agree with measurements of changes in the use of HMLα as the donor, when we perturb the refolding of chromosome III. These results establish refolding of yeast chromosome III as a key driving force in MAT switching and provide an example of a cell regulating the spatial organization of its chromosome so as to direct homology search during recombination.

A novel epigenetic clock for rhesus macaques unveils an association between early life adversity and epigenetic age acceleration.

Because DNA methylation changes reliably with age, machine learning models called epigenetic clocks can estimate an individual's age based on their DNA methylation profile. This epigenetic measure of age can deviate from one's true age, and the difference between the epigenetic age and true age, known as epigenetic age acceleration (EAA), has been found to directly correlate with morbidity and mortality in adults. Emerging evidence suggests that EAA is also associated with aberrant health outcomes in children, making epigenetic clocks useful tools for studying aging and development. We developed two highly accurate epigenetic clocks for the rhesus macaque, utilizing 1,008 blood samples from 690 macaques between 2 days and 23.4 years of age with diverse genetic backgrounds and exposure to environmental conditions. The first clock, which is trained on all samples, achieves a Pearson correlation between true age and predicted age of 0.983 and median absolute error of 0.210 years. To study phenotypes during development, the second clock is optimized for macaques younger than 6 years and achieves a Pearson correlation of 0.974 and a median absolute error of 0.148 years. Using the latter clock, we investigated whether epigenetic aging is affected by early life adversity in the form of infant maltreatment. Our data suggests that maltreatment and increased hair cortisol levels are associated with epigenetic age acceleration right after the period of maltreatment.

Effect of chromosome tethering on nuclear organization in yeast.

Interphase chromosomes in Saccharomyces cerevisiae are tethered to the nuclear envelope at their telomeres and to the spindle pole body (SPB) at their centromeres. Using a polymer model of yeast chromosomes that includes these interactions, we show theoretically that telomere attachment to the nuclear envelope is a major determinant of gene positioning within the nucleus only for genes within 10 kb of the telomeres. We test this prediction by measuring the distance between the SPB and the silent mating locus (HML) on chromosome III in wild-type and mutant yeast strains that contain altered chromosome-tethering interactions. In wild-type yeast cells we find that disruption of the telomere tether does not dramatically change the position of HML with respect to the SPB, in agreement with theoretical predictions. Alternatively, using a mutant strain with a synthetic tether that localizes an HML-proximal site to the nuclear envelope, we find a significant change in the SPB-HML distance, again as predicted by theory. Our study quantifies the importance of tethering at telomeres on the organization of interphase chromosomes in yeast, which has been shown to play a significant role in determining chromosome function such as gene expression and recombination.