Mutational dynamics of a haploid y-specific minisatellite

MSY1 is the only hypervariable minisatellite on the human Y chromosome. Arrays contain 22-114 AT-rich repeat units of 25bp in length, of which eighteen different sequence-variant types have been identified. MSY1 maintains >99% virtual heterozygosity despite the absence of interallelic processes on the haploid and non-recombining Y chromosome. This thesis aimed to determine the types and rates of mutation events that were occurring at MSY1. Single-molecule analysis was used to analyse mutation in the sperm and blood DNA of a single donor. Sperm mutation rate was 2.6%, and mutants were small-scale length changes and isometric mutations that alter the internal structure of arrays but not array length. Overall mutation rate in blood was similar (1.8%), but the spectrum of mutation types was markedly different, suggesting that somatic and germline mutation processes are distinct. Analysis of MSY1 diversity in the framework of the Y phylogeny defined by binary markers allowed inferences to be made about rarer mutation events. These include the generation of novel repeats by recurrent point mutations and conversion events involving a flanking half-repeat (processes with rates of ~10-5 per generation), as well as examples of the rapid spread of specific base substitutions within arrays. The mechanisms underlying this rich variety of mutations could include unequal sister chromatid exchange, replication slippage, synthesis-dependent strand annealing and patch gene conversion. MSY1 repeats are strongly predicted to form hairpins, and this secondary structure is likely to be important in mutation at this unique locus

Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germline

Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germlin

The effects of in utero irradiation on mutation induction and transgenerational instability in mice

Epidemiological evidence suggests that the deleterious effects of prenatal irradiation can manifest during childhood, resulting in an increased risk of leukaemia and solid cancers after birth. However, the mechanisms underlying the long-term effects of foetal irradiation remain poorly understood. This study was designed to analyse the impact of in utero irradiation on mutation rates at expanded simple tandem repeat (ESTR) DNA loci in directly exposed mice and their first-generation (F1) offspring. ESTR mutation frequencies in the germline and somatic tissues of male and female mice irradiated at 12 days of gestation remained highly elevated during adulthood, which was mainly attributed to a significant increase in the frequency of singleton mutations. The prevalence of singleton mutations in directly exposed mice suggests that foetal irradiation results in genomic instability manifested both in utero and during adulthood. The frequency of ESTR mutation in the F1 offspring of prenatally irradiated male mice was equally elevated across all tissues, which suggests that foetal exposure results in transgenerational genomic instability. In contrast, maternal in utero exposure did not affect the F1 stability. Our data imply that the passive erasure of epigenetic marks in the maternal genome can diminish the transgenerational effects of foetal irradiation and therefore provide important clues to the still unknown mechanisms of radiation-induced genomic instability. The results of this study offer a plausible explanation for the effects of in utero irradiation on the risk of leukaemia and solid cancers after birth

Complex germline and somatic mutation processes at a haploid human minisatellite shown by single-molecule analysis.

Mutation at most human minisatellites is driven by complex interallelic processes that give rise to a high degree of length polymorphism and internal structural variation. MSY1, the only highly variable minisatellite on the non-recombining region of the Y chromosome, is constitutively haploid and therefore precluded from interallelic interactions, yet maintains high diversity in both length and structure. To investigate the basis of its mutation processes, an unbiased structural analysis of >500 single-molecule MSY1 PCR products from matched sperm and blood samples from a single donor was undertaken. The overall mutation frequencies in sperm and blood DNAs were not significantly different, at 2.68% and 1.88%, respectively. Sperm DNA showed significantly more length mutants than blood DNA, with mutants in both tissues involving small-scale (1–3 repeat units in a 77 repeat progenitor allele) increases or decreases in repeat block lengths, with no gain or loss bias. Isometric mutations altering structure but not length were found in both tissues, and involved either the apparent shift of a boundary between repeat unit blocks (a ‘boundary switch’) or the conversion of a repeat within a block to a different repeat type (‘modular structure’ mutant). There was a significant excess of boundary switch mutants and deficit of modular structure mutants in sperm. A comparison of mutant structures with phylogenetically matched alleles in population samples showed that alleles with structures resembling the blood mutants were unlikely to arise in populations. Mutation seems likely to involve gene conversion via synthesis-dependent strand annealing, and the blood-sperm differences may reflect more relaxed constraint on sister chromatid alignment in blood