Insights into the molecular orchestration of DNA double-strand break signaling and repair proteins: A functional evaluation of the MutS homolog hMSH5

Cells are exquisitely adapted to cope with DNA damage, possessing the molecular machinery to accommodate the intricacies of DNA damage sensing, signaling, and repair pathways. Of the numerous forms of DNA damage, the DNA double-strand break (DSB) is considered the most lethal. Accordingly, numerous studies have established deficiencies in DSB repair to be directly associated with chromosome instability -- a hallmark of tumor cells. Though a member of the MutS homolog family of mismatch repair proteins, the human MutS homolog 5 (hMSH5) has been implicated in the repair of programmed DSBs during meiotic homologous recombination (HR). Specifically, meiotic studies indicate hMSH5 may facilitate homology searching and appropriate homologous chromosome alignment, leading to crossover formation between homologous chromosomes. The thesis research presented here aims to characterize the role and regulation of hMSH5. To this end, DNA DSB-induced hMSH5 recruitment and involvement in DSB repair was comprehensibly characterized among several other repair factors. hMSH5 and subsequently hMSH4 assembly at an inducible DSB was dependent on hMRE11 and hRad51 proteins, as well c-Abl mediated tyrosine phosphorylation of hMSH5 at Y742. Recombinational repair analysis indicate hMSH5 to promote both crossover and non-crossover HR outcomes, in which deficiencies in hMSH5 recruitment significantly disrupted HR and sensitized cells to the chemotherapeutic cisplatin.;Beyond roles in DNA repair, numerous cellular repair factors also function to coordinate the precise interrelationships between DNA repair with cell cycle arrest and/or under extensive DNA damage, apoptotic signaling. Considering the fundamental role of c-Abl in tailoring cellular responses to DNA damage and as such the necessity to tightly regulate c-Abl catalytic activity, the hMSH5-c-Abl interaction was additionally examined for DNA damage signaling potential through c-Abl activation. Analysis of hMSH5, and the polymorphic variant hMSH5P29S, indicates hMSH5 and more so hMSH5 P29S to stimulate c-Abl activation, the stabilization of p73, and to promote heightened ionizing radiation induced apoptosis. Taken together, the dynamic interplay between hMSH5 and c-Abl may critically function in deciding cell fate to DNA insults that stimulate c-Abl activity, through activation of either DNA repair or apoptosis.