High-Resolution Genome-Wide Analysis of Irradiated (UV and γ-Rays) Diploid Yeast Cells Reveals a High Frequency of Genomic Loss of Heterozygosity (LOH) Events

In diploid eukaryotes, repair of double-stranded DNA breaks by homologous recombination often leads to loss of heterozygosity (LOH). Most previous studies of mitotic recombination in Saccharomyces cerevisiae have focused on a single chromosome or a single region of one chromosome at which LOH events can be selected. In this study, we used two techniques (single-nucleotide polymorphism microarrays and high-throughput DNA sequencing) to examine genome-wide LOH in a diploid yeast strain at a resolution averaging 1 kb. We examined both selected LOH events on chromosome V and unselected events throughout the genome in untreated cells and in cells treated with either γ-radiation or ultraviolet (UV) radiation. Our analysis shows the following: (1) spontaneous and damage-induced mitotic gene conversion tracts are more than three times larger than meiotic conversion tracts, and conversion tracts associated with crossovers are usually longer and more complex than those unassociated with crossovers; (2) most of the crossovers and conversions reflect the repair of two sister chromatids broken at the same position; and (3) both UV and γ-radiation efficiently induce LOH at doses of radiation that cause no significant loss of viability. Using high-throughput DNA sequencing, we also detected new mutations induced by γ-rays and UV. To our knowledge, our study represents the first high-resolution genome-wide analysis of DNA damage-induced LOH events performed in any eukaryote

Molecular Poltergeists: Mitochondrial DNA Copies (numts) in Sequenced Nuclear Genomes

The natural transfer of DNA from mitochondria to the nucleus generates nuclear copies of mitochondrial DNA (numts) and is an ongoing evolutionary process, as genome sequences attest. In humans, five different numts cause genetic disease and a dozen human loci are polymorphic for the presence of numts, underscoring the rapid rate at which mitochondrial sequences reach the nucleus over evolutionary time. In the laboratory and in nature, numts enter the nuclear DNA via non-homolgous end joining (NHEJ) at double-strand breaks (DSBs). The frequency of numt insertions among 85 sequenced eukaryotic genomes reveal that numt content is strongly correlated with genome size, suggesting that the numt insertion rate might be limited by DSB frequency. Polymorphic numts in humans link maternally inherited mitochondrial genotypes to nuclear DNA haplotypes during the past, offering new opportunities to associate nuclear markers with mitochondrial markers back in time

Numt-Mediated Double-Strand Break Repair Mitigates Deletions during Primate Genome Evolution

Non-homologous end joining (NHEJ) is the major mechanism of double-strand break repair (DSBR) in mammalian cells. NHEJ has traditionally been inferred from experimental systems involving induced double strand breaks (DSBs). Whether or not the spectrum of repair events observed in experimental NHEJ reflects the repair of natural breaks by NHEJ during chromosomal evolution is an unresolved issue. In primate phylogeny, nuclear DNA sequences of mitochondrial origin, numts, are inserted into naturally occurring chromosomal breaks via NHEJ. Thus, numt integration sites harbor evidence for the mechanisms that act on the genome over evolutionary timescales. We have identified 35 and 55 lineage-specific numts in the human and chimpanzee genomes, respectively, using the rhesus monkey genome as an outgroup. One hundred and fifty two numt-chromosome fusion points were classified based on their repair patterns. Repair involving microhomology and repair leading to nucleotide additions were detected. These repair patterns are within the experimentally determined spectrum of classical NHEJ, suggesting that information from experimental systems is representative of broader genetic loci and end configurations. However, in incompatible DSBR events, small deletions always occur, whereas in 54% of numt integration events examined, no deletions were detected. Numts show a statistically significant reduction in deletion frequency, even in comparison to DSBR involving filler DNA. Therefore, numts show a unique mechanism of integration via NHEJ. Since the deletion frequency during numt insertion is low, native overhangs of chromosome breaks are preserved, allowing us to determine that 24% of the analyzed breaks are cohesive with overhangs of up to 11 bases. These data represent, to the best of our knowledge, the most comprehensive description of the structure of naturally occurring DSBs. We suggest a model in which the sealing of DSBs by numts, and probably by other filler DNA, prevents nuclear processing of DSBs that could result in deleterious repair

Mosaic mitochondrial-plastid insertions into the nuclear genome show evidence of both non-homologous end joining and homologous recombination

Abstract Background Mitochondrial and plastid DNA fragments are continuously transferred into eukaryotic nuclear genomes, giving rise to nuclear copies of mitochondrial DNA (numts) and nuclear copies of plastid DNA (nupts). Numts and nupts are classified as simple if they are composed of a single organelle fragment or as complex if they are composed of multiple fragments. Mosaic insertions are complex insertions composed of fragments of both mitochondrial and plastid DNA. Simple numts and nupts in eukaryotes have been extensively studied, their mechanism of insertion involves non-homologous end joining (NHEJ). Mosaic insertions have been less well-studied and their mechanisms of integration are unknown. Results Here we estimated the number of nuclear mosaic insertions (numins) in nine plant genomes. We show that numins compose up to 10% of the total nuclear insertions of organelle DNA in these plant genomes. The NHEJ hallmarks typical for numts and nupts were also identified in mosaic insertions. However, the number of identified insertions that integrated via NHEJ mechanism is underestimated, as NHEJ signatures are conserved only in recent insertions and mutationally eroded in older ones. A few complex insertions show signatures of long homology that cannot be attributed to NHEJ, a novel observation that implicates gene conversion or single strand annealing mechanisms in organelle nuclear insertions. Conclusions The common NHEJ signature that was identified here reveals that, in plant cells, mitochondria and plastid fragments in numins must meet during or prior to integration into the nuclear genome

Whole Genome Sequence Analysis of Mutations Accumulated in rad27Δ Yeast Strains with Defects in the Processing of Okazaki Fragments Indicates Template-Switching Events

Okazaki fragments that are formed during lagging strand DNA synthesis include an initiating primer consisting of both RNA and DNA. The RNA fragment must be removed before the fragments are joined. In Saccharomyces cerevisiae, a key player in this process is the structure-specific flap endonuclease, Rad27p (human homolog FEN1). To obtain a genomic view of the mutational consequence of loss of RAD27, a S. cerevisiae rad27Δ strain was subcultured for 25 generations and sequenced using Illumina paired-end sequencing. Out of the 455 changes observed in 10 colonies isolated the two most common types of events were insertions or deletions (INDELs) in simple sequence repeats (SSRs) and INDELs mediated by short direct repeats. Surprisingly, we also detected a previously neglected class of 21 template-switching events. These events were presumably generated by quasi-palindrome to palindrome correction, as well as palindrome elongation. The formation of these events is best explained by folding back of the stalled nascent strand and resumption of DNA synthesis using the same nascent strand as a template. Evidence of quasi-palindrome to palindrome correction that could be generated by template switching appears also in yeast genome evolution. Out of the 455 events, 55 events appeared in multiple isolates; further analysis indicates that these loci are mutational hotspots. Since Rad27 acts on the lagging strand when the leading strand should not contain any gaps, we propose a mechanism favoring intramolecular strand switching over an intermolecular mechanism. We note that our results open new ways of understanding template switching that occurs during genome instability and evolution

Dating <i>numt</i> insertion.

<p>(A) Dating <i>numt</i> insertion based on a mitochondrial phylogenetic tree (black branches). An arrow indicates time of insertion and the <i>numt</i> branch is shown in red. The methodology can be used only in species where the mitochondrial rate of evolution is lower than the nuclear rate of evolution (e.g., mammals but not plants) and when the <i>numts</i> are long enough (>1 kb) to carry enough evolutionary signal. (B) Dating <i>numt</i> insertion based on patterns of presence and absence on a phylogeny. Few nuclear genomes and their genome alignment are used to identify <i>numt</i> insertions. Species that share the descendant from the common ancestor where the transfer occurred include the <i>numts</i> (red rectangle) whereas this <i>numt</i> is missing in the others.</p

Blast analysis of 85 mitochondria against their nuclear genomes (BlastN, e-score = 0.0001).

<p>For each organism the number of BLAST hits as well as the unique number of bases in genomes is given (i.e. a base in the genome that has a BLAST hit to two repetitive mitochondria pieces it is count only once in <i>numt</i> content). Other available <i>numt</i> estimates are indicated with their references, where the corresponding search parameters are given.</p