Biochemistry of a Mutant, Cancer-causing DNA Licensing Protein MCM4
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Chromosomal instability is a hallmark of cancer cells and impaired DNA replication is a major cause of chromosome instability. This thesis investigates the mutant allele Chaos3 (chromosome aberrations occurring spontaneously 3) isolated in a N-ethyl- N-nitrosourea (ENU) mutagenesis screen of mice for chromosome instability. This mutation causes high levels of chromosome instability. Over 80% of mutant female homozygous mice develop mammary adenocarcinomas with a mean latency of 12 months. The Chaos3 mutation occurs in the Mcm4 gene (minichromosome maintenance 4) and causes a single amino acid change (F345I) in a highly conserved region. Mcm4 encodes the MCM4 subunit of the hetero-hexameric MCM2-7 complex. MCM2-7 is a DNA replicative helicase. During DNA replication, MCM2-7 is loaded on the replication origins as double hexamers. The goal of this study was to characterize the biochemical consequence of the point mutation, focusing on protein interactions measured by co-immunoprecipitation. Here, we report that the Chaos3 mutation causes a dramatic decrease of MCM4-MCM6 interaction and a slight decrease of MCM4-MCM7 interaction. This finding suggests that the loss of interaction might cause structural instability of the replicative machinery, leading to increased number of stalled replication forks and chromosome segregation defects. Another finding was identifying an interaction between MCM4 and HSP70.
dissertation or thesis