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

Shanks ME, May CA, Dubrova YE, Balaresque P, Rosser ZH, Adams SM, Jobling MA - Mutat. Res. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, UK.

ABSTRACT
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.

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Synthesis-dependent strand annealing as a candidate mechanism for MSY1 mutation. Boundary switch mutant arising from sister chromatids misaligned by a single-repeat-unit. Modular structural mutant arising from sister chromatids misaligned by two repeat units. Example of a length mutant arising from aligned sister chromatids. Open arrows indicate repeat units (black: type 3; grey: type 4); dashed arrows indicate DNA synthesis.
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fig5: Synthesis-dependent strand annealing as a candidate mechanism for MSY1 mutation. Boundary switch mutant arising from sister chromatids misaligned by a single-repeat-unit. Modular structural mutant arising from sister chromatids misaligned by two repeat units. Example of a length mutant arising from aligned sister chromatids. Open arrows indicate repeat units (black: type 3; grey: type 4); dashed arrows indicate DNA synthesis.

Mentions: Synthesis-dependent strand annealing (SDSA) [27] is a gene conversion mechanism that has been proposed to explain mutation at GC-rich autosomal minisatellites, including MS32, MS205 and CEB1 [3,6,17]. This mechanism, acting between sister chromatids, could be responsible for the more complex events observed in MSY1 mutation (Fig. 5). The first step is a double-strand break – a lesion that might be promoted by replication fork stalling [28], possibly through the formation of cruciform structures within the array. Following resection, a strand from one chromatid invades the other, thereby creating a D-loop. After DNA synthesis and resolution, the result is the unidirectional transfer of sequence information from one chromatid to another. The outcome, in terms of array change, depends on the initial register of alignment of the sister chromatids. If they are misaligned by one repeat unit (Fig. 5(a)), then a boundary switch mutation can result; if misalignment is by more than one repeat unit, then a modular structural change can occur (Fig. 5(b)). The general observation that modular structural mutants involve repeat type switching of only single-repeat-units suggests that the scale of these conversion events must be restricted (≤25 bp). The position of the converted repeat is dependent on the extent of sister chromatid misalignment; the difference between blood and sperm DNA can then be interpreted as a relaxation of the alignment in the former, allowing conversion events to occur deeper within the blocks of type 3 and type 4 repeats. SDSA can also be invoked to explain length change mutants (Fig. 5(c)). Differences, discussed above, between MSY1 and MS32/CEB1 in germline and somatic intra-allelic mutation behaviour may indicate that the relaxation of sister chromatid exchange, we infer in blood, may not be a general phenomenon, but region- or locus-specific.


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

Shanks ME, May CA, Dubrova YE, Balaresque P, Rosser ZH, Adams SM, Jobling MA - Mutat. Res. (2008)

Synthesis-dependent strand annealing as a candidate mechanism for MSY1 mutation. Boundary switch mutant arising from sister chromatids misaligned by a single-repeat-unit. Modular structural mutant arising from sister chromatids misaligned by two repeat units. Example of a length mutant arising from aligned sister chromatids. Open arrows indicate repeat units (black: type 3; grey: type 4); dashed arrows indicate DNA synthesis.
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fig5: Synthesis-dependent strand annealing as a candidate mechanism for MSY1 mutation. Boundary switch mutant arising from sister chromatids misaligned by a single-repeat-unit. Modular structural mutant arising from sister chromatids misaligned by two repeat units. Example of a length mutant arising from aligned sister chromatids. Open arrows indicate repeat units (black: type 3; grey: type 4); dashed arrows indicate DNA synthesis.
Mentions: Synthesis-dependent strand annealing (SDSA) [27] is a gene conversion mechanism that has been proposed to explain mutation at GC-rich autosomal minisatellites, including MS32, MS205 and CEB1 [3,6,17]. This mechanism, acting between sister chromatids, could be responsible for the more complex events observed in MSY1 mutation (Fig. 5). The first step is a double-strand break – a lesion that might be promoted by replication fork stalling [28], possibly through the formation of cruciform structures within the array. Following resection, a strand from one chromatid invades the other, thereby creating a D-loop. After DNA synthesis and resolution, the result is the unidirectional transfer of sequence information from one chromatid to another. The outcome, in terms of array change, depends on the initial register of alignment of the sister chromatids. If they are misaligned by one repeat unit (Fig. 5(a)), then a boundary switch mutation can result; if misalignment is by more than one repeat unit, then a modular structural change can occur (Fig. 5(b)). The general observation that modular structural mutants involve repeat type switching of only single-repeat-units suggests that the scale of these conversion events must be restricted (≤25 bp). The position of the converted repeat is dependent on the extent of sister chromatid misalignment; the difference between blood and sperm DNA can then be interpreted as a relaxation of the alignment in the former, allowing conversion events to occur deeper within the blocks of type 3 and type 4 repeats. SDSA can also be invoked to explain length change mutants (Fig. 5(c)). Differences, discussed above, between MSY1 and MS32/CEB1 in germline and somatic intra-allelic mutation behaviour may indicate that the relaxation of sister chromatid exchange, we infer in blood, may not be a general phenomenon, but region- or locus-specific.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Leicester, Leicester, UK.

ABSTRACT
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.

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