%0 Generic
%9 Doctoral Dissertation
%A Jeffries, Elizabeth Paladin
%D 2015
%F pittir:25446
%K DNA Replication, homologous recombination, alternative splicing, premature ovarian failure, meiosis
%T HsMCM8 and HsMCM9: Essential for Double-Strand Break Repair and Normal Ovarian Function
%U http://d-scholarship-dev.library.pitt.edu/25446/
%X The minichromosome maintenance (MCM) family of proteins is conserved from archaea to humans, and its members have roles in initiating DNA replication. MCM8 and MCM9 are minimally characterized members of the eukaryotic MCM family that associate with one another and both contain conserved ATP binding and hydrolysis motifs. The MCM8-9 complex participates in repair of DNA double-strand breaks by homologous recombination, and MCM8 is implicated in meiotic recombination. We identified a novel alternatively spliced isoform of HsMCM9 that results in a medium length protein product (MCM9M) that eliminates a C-terminal extension of the fully spliced product (MCM9L). Quantitative real-time reverse transcriptase PCR (qRT-PCR) of the relative mRNA isoform abundances across cell lines reveals MCM9L transcript is more abundant than MCM9M. The expression of both isoforms is cell cycle regulated, as they are most abundant in S-phase. Wild-type MCM9L forms DNA damage-dependent nuclear foci, while MCM9M is cytoplasmic and MCM9Cterm is diffuse throughout the nucleus. We have identified and verified a putative nuclear localization signal (NLS), and Rad51-interacting motif (BRCv) in the C-terminus of MCM9. GFP-tagged MCM9 NLS- is solely cytoplasmic, GFP-tagged MCM9 BRCv- is diffuse throughout the nucleus. A combination of SNP arrays, comparative genomic hybridization arrays, and whole-exome sequencing analyses identified homozygous pathogenic variants in MCM8 (MCM8 c.446C>G; p.P149R) and MCM9 (MCM9 c.1732.2T>C and  MCM9 c.394C>T; p.R132*), all located within regions of homozygosity in women afflicted by premature ovarian failure (POF). The MCM9 c.1732.2T>C variant alters a splice donor site, resulting in abnormal alternative splicing and truncated forms of MCM9 that are unable to be recruited to sites of DNA damage. In the second family, MCM9 c.394C>T (p.R132*) results in a predicted loss of functional MCM9. Compared with fibroblasts from unaffected family members, chromosomal break repair was deficient in fibroblasts from all affected individuals, likely due to inhibited recruitment of mutated MCM8 or MCM9 to sites of DNA damage. Our cumulative results suggest that MCM8-9 have evolved to act late in both mitotic recombination pathways to aid in crossover migration and/or strand resolution.