Conservative Inheritance of Newly Synthesized DNA in Double-Strand Break-Induced Gene Conversion

To distinguish among possible mechanisms of repair of a double-strand break (DSB) by gene conversion in budding yeast, Saccharomyces cerevisiae, we employed isotope density transfer to analyze budding yeast mating type (MAT) gene switching in G(2)/M-arrested cells. Both of the newly synthesized DNA strands created during gene conversion are found at the repaired locus, leaving the donor unchanged. These results support suggestions that mitotic DSBs are primarily repaired by a synthesis-dependent strand-annealing mechanism. We also show that the proportion of crossing-over associated with DSB-induced ectopic recombination is not affected by the presence of nonhomologous sequences at one or both ends of the DSB or the presence of additional sequences that must be copied from the donor

Heritable Change Caused by Transient Transcription Errors

<div><p>Transmission of cellular identity relies on the faithful transfer of information from the mother to the daughter cell. This process includes accurate replication of the DNA, but also the correct propagation of regulatory programs responsible for cellular identity. Errors in DNA replication (mutations) and protein conformation (prions) can trigger stable phenotypic changes and cause human disease, yet the ability of transient transcriptional errors to produce heritable phenotypic change (‘epimutations’) remains an open question. Here, we demonstrate that transcriptional errors made specifically in the mRNA encoding a transcription factor can promote heritable phenotypic change by reprogramming a transcriptional network, without altering DNA. We have harnessed the classical bistable switch in the <i>lac</i> operon, a memory-module, to capture the consequences of transient transcription errors in living <i>Escherichia coli</i> cells. We engineered an error-prone transcription sequence (A₉ run) in the gene encoding the <i>lac</i> repressor and show that this ‘slippery’ sequence directly increases epigenetic switching, not mutation in the cell population. Therefore, one altered transcript within a multi-generational series of many error-free transcripts can cause long-term phenotypic consequences. Thus, like DNA mutations, transcriptional epimutations can instigate heritable changes that increase phenotypic diversity, which drives both evolution and disease.</p></div

Phenotypic consequences from errors in information transfer in a cellular lineage.

<p>Wild-type genes (black parallel lines) make wild-type transcripts (blue wavy lines) make wild-type functional proteins (blue circles); mutant genes make mutant transcripts (red crosses) make mutant proteins (red circles); protein mis-folding can trigger phenotypic change by changing protein conformation to the prion state (red triangle) that can self-perpetuate by templating the aberrant conformation with nascent native proteins (blue triangles). From wild-type genes can also come altered mRNA (epimutation) making altered proteins that can perturb transcriptional networks in a nonlinear manner generating a heritable phenotypic change (red arrows) from a transient stochastic error in information transfer. In this case no trace of the error will remain in the lineage after the phenotypic change as indicated: while change through mutation will retain evidence of the original stochastic error in the progeny cell (mutant DNA, mutant RNA and mutant protein), change through epimutation will retain no evidence of the original stochastic error in the progeny cell (WT DNA, WT RNA and WT protein). Errors in DNA and RNA synthesis occur at rates of, very roughly, 10⁻⁹ and 10⁻⁵ errors per residue, respectively <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003595#pgen.1003595-Ninio1" target="_blank">[48]</a>; yeast cells in the non-prion [<i>psi</i>⁻] state spontaneously switch <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003595#pgen.1003595-Halfmann1" target="_blank">[43]</a> to [<i>PSI</i>⁺] at a frequency of 10⁻⁶; the great majority of cells will not have sustained any errors in information transfer.</p

The error-prone A₉ run in the <i>lacI</i> transcript increases stochastic phenotypic switching.

<p>(A) Representative flow cytometry GFP fluorescence histogram series of A₉ and A₅GA₃<i>lacI</i> cells that were originally ON (green histograms) or OFF (red histograms) were sub-cultured and grown in media containing various concentrations of TMG indicated on the vertical axis (10⁴ cells interrogated). Below 5 µM TMG and above 20 µM TMG, the previous history of the cell (ON or OFF) does not affect the current state of the cell; between these TMG concentrations the system exhibits hysteresis. The shaded area highlights the maintenance concentration of 9 µM TMG for these strains. (B) Cells that were originally ON or OFF were sub-cultured and grown in media containing various concentrations of TMG, as above. Each value is the average ± SD from 5 to 15 independent cultures. The shaded area highlights the maintenance concentration of 9 µM TMG for these strains. (C) OFF A₅GA₃<i>lacI</i> cells (red histograms) and A₉<i>lacI</i> cells (blue histograms) were diluted and grown in media containing 9 µM TMG. After 42 h growth, flow cytometry was performed to determine the frequency of epigenetically ON cells in 20 independent cultures of each strain; the A₅GA₃ histograms are superimposed over the A₉ histograms (10⁴ cells interrogated). (D) The A₉ epigenetic-switch frequency is significantly increased over the A₅GA₃ value (Mann-Whitney Rank Sum Test, <i>p</i><0.001).</p

Novel system to study the consequences of error-prone transcription sequences.

<p>(A) Under maintenance conditions, the <i>lac</i> operon is OFF (indicated by the solid red line) and the inducer TMG remains extracellular; stochastic events that lead to a transient derepression of the <i>lac</i> operon will initiate an autocatalytic positive-feedback response (indicated by solid blue lines). The box highlights the first three codons of the wild-type <i>lac</i> repressor gene and the Lys-Lys additions encoded by the A₉ and A₅GA₃<i>lacI</i> alleles (in red). (B) The <i>lacI</i> A₉ and A₅GA₃ Lys-Lys N-terminal addition alleles encode functional <i>lac</i> repressors. Representative flow cytometry analyses measuring GFP fluorescence of OFF and ON populations of wild-type (red, brown), A₉ (blue, light blue) and A₅GA₃ (green, violet) <i>lacI</i> cells produce identical histograms; 10⁴ cells of each strain were interrogated. (C) Forward <i>lacI</i>⁺→<i>lacI</i>⁻ mutation frequencies. No significant difference in mutation frequency between the A₉ and A₅GA₃ strains is observed (Mann-Whitney Rank Sum Test, <i>p</i> = 0.23). The wild-type strain is added for comparison.</p

Transcription errors, not translational frameshifting, at the <i>lacI</i> A₉ sequence influences stochastic switching.

<p>(A) Stochastic phenotypic switching is significantly increased when the error-prone A₉ run is in a transcription fidelity-deficient background (Δ<i>greA</i> Δ<i>greB</i> cells). OFF Δ<i>greAB</i> A₅GA₃<i>lacI</i> cells (red histograms) and Δ<i>greAB</i> A₉<i>lacI</i> cells (blue histograms) were diluted and grown in media containing 9 µM TMG. After 42 h growth, flow cytometry was performed to determine the frequency of epigenetically ON cells in 17–19 independent cultures of each strain; the histograms from the Δ<i>greAB</i> A₅GA₃<i>lacI</i> cultures are superimposed over the histograms from the Δ<i>greAB</i> A₉<i>lacI</i> cultures; each histogram represents the interrogation of 10⁴ cells. (B) The median for the Δ<i>greAB</i> A₉<i>lacI</i> strain is significantly different from the Δ<i>greAB</i> A₅GA₃<i>lacI</i> value (Mann-Whitney Rank Sum Test, <i>p</i><0.001). (C) To model translation frameshifting in our system we have created merodiploids that provide a 10-fold excess of wild-type transcripts over ±1 frameshift transcripts (as modeled by the A₈ and A₁₀<i>lacI</i> alleles). Therefore, the ratio of wild-type transcript over frameshifted transcript, at the level of transcription (10∶1), will be a very conservative approximation of the situation that would arise if during the translation of one A₉ transcript, one translational frameshift event would occur (20∶1 wild-type sub-units over frameshifted sub-units). The wild-type repressor allele is completely dominant over the frameshifted repressor alleles: left panel, the <i>lacI</i> allele strains without the F′; right panel, the <i>lacI</i> allele strains with the F′ overproducing wild-type <i>lacI</i>. The glucose minimal plates include Xgal (40 µg/ml) and tetracycline (Tet, 12.5 µg/ml), as indicated beneath the plate. Tet is used to maintain the F′ in the cell. (D) Quantitative measurement of the phenotype observed in (C). The level of β-galactosidase in all four strains is comparable and does not exceed 1 Miller unit, which is the basal β-galactosidase of uninduced <i>E. coli</i> cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003595#pgen.1003595-Miller1" target="_blank">[33]</a>; the average ± SD for three independent cultures is shown. (E) A −1 translational frameshifting event at the A₉ sequence would cause translation to terminate at codon 4/5 (green line denotes wild-type protein; gray line denotes frameshifted protein; red X denotes translation termination; blue line denotes translation reinitiation protein; the A₉ transcript is shown as a black line with the GUG start codon in green letters and the UGA stop codon in red letters; the protein domain structure is indicated above the translation products). Therefore, no functional <i>lac</i> repressor sub-unit could be produced; however, it has been shown that a dominant-negative sub-unit could be produced by translational reinitiation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003595#pgen.1003595-Platt1" target="_blank">[61]</a>. Reinitiation could occur at codons 23, 24, 38 or 42 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003595#pgen.1003595-Steege1" target="_blank">[34]</a>, producing repressor sub-units lacking the DNA-binding domain but the core aggregation domain would be intact and able to bind and interfere with wild-type sub-unit function. Therefore, there is the possibility that one −1 translational frameshifting event would not only decrease the net total of repressor sub-units by one, but might also decrease the cell's net <i>lac</i> repressors by one, since it has been shown that one dominant-negative sub-unit with three wild-type sub-units may abolish the function of the tetrameric <i>lac</i> repressor <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003595#pgen.1003595-MllerHill1" target="_blank">[42]</a>. A +1 translational frameshifting event at the A₉ sequence (generating a A₁₀ transcript) would cause translation to terminate at codon 83/84 and would therefore only result in the net decrease of one repressor sub-unit in the cell, since no dominant-negative sub-unit can be made this far into the core domain. When the wild-type sub-unit is made at 10-fold the level of ±1 transcription frameshift events (and ±1 translation protein products), the wild-type sub-units dominate and the <i>lac</i> operon is repressed (as seen in the Xgal Tet F′ <i>lacI^q</i> plate in (C) and therefore any net decrease by one translation frameshift event is negligible when compared to the net decrease in repressor sub-units due to a transcription error. When a transcription error occurs at the A₉ sequence, all the nascent <i>lac</i> repressor sub-units will be non-functional (and/or dominant-negative); when a translational frameshift occurs at the A₉ sequence, less than 1/10 of all nascent <i>lac</i> repressor sub-units will be non-functional (and/or dominant-negative).</p

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

Eukaryotes possess mechanisms to limit crossing over during homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases—Srs2 and Sgs1—that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks