Alison J. Rattray

Elevated Mutation Rate during Meiosis in Saccharomyces cerevisiae

Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960′s Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called “the meiotic effect.” Here we use a forward mutation reporter (CAN1 HIS3) placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 –fold) correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts). Polζ appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.

GAP-Seq: a method for identification of DNA palindromes

BackgroundClosely spaced long inverted repeats, also known as DNA palindromes, can undergo intrastrand annealing to form DNA hairpins. The ability to form these hairpins results in genome instability, difficulties in maintaining clones in Escherichia coli and major problems for most DNA sequencing approaches. Because of their role in genomic instability and gene amplification in some human cancers, it is important to develop systematic approaches to detect and characterize DNA palindromes.ResultsWe developed a new protocol to identify palindromes that couples the S1 nuclease treated Cot0 DNA (GAPF) with high-throughput sequencing (GAP-Seq). Unlike earlier protocols, it does not involve restriction enzymatic digestion prior to DNA snap-back thereby preserving longer DNA sequences. It also indicates the location of the novel junction, which can then be recovered. Using MCF-7 breast cancer cell line as the proof-of-principle analysis, we have identified 35 palindrome candidates and physically characterized the top 5 candidates and their junctions. Because this protocol eliminates many of the false positives that plague earlier techniques, we have improved palindrome identification.ConclusionsThe GAP-Seq approach underscores the importance of developing new tools for identifying and characterizing palindromes, and provides a new strategy to systematically assess palindromes in genomes. It will be useful for studying human cancers and other diseases associated with palindromes.

A Cre Transcription Fidelity Reporter Identifies GreA as a Major RNA Proofreading Factor in Escherichia coli

We made a coupled genetic reporter that detects rare transcription misincorporation errors to measure RNA polymerase transcription fidelity in Escherichia coli. Using this reporter, we demonstrated in vivo that the transcript cleavage factor GreA, but not GreB, is essential for proofreading of a transcription error where a riboA has been misincorporated instead of a riboG. A greA mutant strain had more than a 100-fold increase in transcription errors relative to wild-type or a greB mutant. However, overexpression of GreB in ΔgreA cells reduced the misincorporation errors to wild-type levels, demonstrating that GreB at high concentration could substitute for GreA in RNA proofreading activity in vivo.