Link to the University of Pittsburgh Homepage
Link to the University Library System Homepage Link to the Contact Us Form

HsMCM8 and HsMCM9: Essential for Double-Strand Break Repair and Normal Ovarian Function

Jeffries, Elizabeth Paladin (2015) HsMCM8 and HsMCM9: Essential for Double-Strand Break Repair and Normal Ovarian Function. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

Primary Text

Download (6MB)


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.


Social Networking:
Share |


Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Jeffries, Elizabeth Paladinecp13@pitt.eduECP13
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee CoChairTrakselis, Michael A.mtraksel@pitt.eduMTRAKSEL
Committee CoChairHorne, Sethhorne@pitt.eduHORNE
Committee MemberLiu, Xinyuxinyuliu@pitt.eduXINYULIU
Committee MemberRajkovic, Aleksandarrajkovic@upmc.eduALR110
Date: 24 September 2015
Date Type: Publication
Defense Date: 4 May 2015
Approval Date: 24 September 2015
Submission Date: 20 June 2015
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 144
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: DNA Replication, homologous recombination, alternative splicing, premature ovarian failure, meiosis
Date Deposited: 24 Sep 2015 23:38
Last Modified: 24 Sep 2017 05:15


Monthly Views for the past 3 years

Plum Analytics

Actions (login required)

View Item View Item