Germline minisatellite instability in the presence and absence of MLH1, a mismatch repair protein

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Perustieteiden korkeakoulu | Master's thesis
Computational Systems Biology
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Master's Degree Programme in Computational and Systems Biology (euSYSBIO)
69 + 8
MLH1 (MutL homolog 1) is a mismatch repair protein that can form heterodimer with MLH3 in meiosis. A functional MLH1-MLH3 heterodimer is essential for crossover recombination. Not much is known about the effect of loss of MLH1 function on mini satellite instability. The aim of this project is to investigate mini satellite instability in germ line cells of wild-type and Mlh1-/- male mice of the C57BL/6 strain, with a particular focus on mini satellite locus Prdm9 (a meiotic recombination protein in metazoans). An attempt is made to find mini satellites that function as potential DNA binding sites for PRDM9 and mini satellite instability analysis is also performed for one of them (Ms-X165.369). Prdm9 locus is compared to a known stable and an unstable mini satellite, Ms-X165.369 and two characterized microsatellites. Spermatocytes were isolated from mouse testis based on their size, and DNA was extracted. As a control, DNA extracted from somatic tissue was used. Small-pool PCR followed by Southern blotting was used to investigate and compare stability of the different mini satellite loci. In silico analysis was conducted to predict DNA binding motifs and DNA binding affinities for newly identified Prdm9 variants. We found germ line instability at Prdm9 locus to be higher in Mlh1-/- mouse than in wild type mouse, with germ line instability of 16.6% and 1.4% per gamete respectively. No somatic mutation was observed at Prdm9 locus for both the genotype. At Ms-X165.369 locus, neither germ line nor somatic mutation was observed in either of the genotype. All the PRDM9 variants were predicted to have different DNA binding motifs. DNA binding affinity of PRDM9 variants to Ms-X165.369 was predicted to increase by at most 39% and decrease by at least 51.5% and to the progenitor PRDM9's DNA binding motif by at least 68%, compared to the progenitor PRDM9. Our results show that MLH1 is required for germ line mini satellite stability.
Rousu, Juho
Thesis advisor
Kauppi, Liisa
germline minisatellite instability, Prdm9, Ms-X165.369, mismatch repair, MLH1, single-molecule PCR
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