Elucidation of the structure-function relationship of the Saccharomyces cerevisiae HORMA domain-containing proteins Hop1 and Rev7

Abstract

DNA-based transactions like replication, repair, recombination and transcription are central to survival, inheritance and evolution. Not surprisingly, these mechanisms are tightly regulated by a myriad of protein-protein, protein-nucleic acid, protein-ligand associations and enzymatic functions. This thesis revolves around elucidating the structure-function relationships of two important HORMA-domain containing proteins Hop1 and Rev7, which function at the very core of these mechanisms. During meiosis, budding yeast Hop1 protein supports the synaptonemal complex, promoting synapsis between homologous chromosomes, double-strand break formation, and inter-homolog recombination. The first part of this thesis reports the unexpected discovery and characterization of DNA-independent ATPase activity of Hop1. Molecular threading and in silico simulation studies uncovered a putative ATP-binding pocket of Hop1 which harbours amino acid residues from both the N- and the C-terminal region of the protein. Specifically, mutation of lysine 65 and asparagine 67 residues in the ATP-binding pocket abrogated both ATP-binding and hydrolysis potential of Hop1 protein, and lead to reduced affinity towards Holliday Junction DNA. While mutations in the ATP binding pocket do not affect meiotic progression or spore viability, crossover frequencies are moderately enhanced in these strains. In agreement with DNA-binding assays, high-resolution calibrated ChIP analyses showed that ATPase-deficient mutant of Hop1 exhibits reduced axis association as compared to the wild-type protein. Taken together, our biochemical, biophysical, in silico and genetics approaches suggest that Hop1 harbours an intrinsic ATPase activity which is critical to maintain wild-type levels of crossing over and facilitates DNA binding. A suite of studies in cancer cell lines established role of Rev7 in regulating DNA repair pathway choice as a component of the conserved tetrameric Shieldin complex. In the absence of a functional homologous recombination (HR) pathway (such as in brca1-/- cells), loss of Rev7 leads to PARP inhibitor resistance and cell survival, suggesting HR restoration. In the absence of Shieldin subunits in budding yeast, we explored the biochemical properties and functions of S. cerevisiae HORMA domain-containing protein Rev7 in regulating DNA repair and recombination. In line with this, classical genetic approaches and plasmid-chromosome recombination assays showed that loss of Rev7 leads to elevated recombination levels in S. cerevisiae cells, suggesting that it indeed functions as an anti-resection factor in this context. Mechanistically, we found that Rev7 interacts with the individual MRX subunits (Mre11-Rad50-Xrs2) in vitro and in vivo; and adversely affects Mre11 nuclease and Rad50 ATPase activities. Plasmid-based and chromosomal end-joining assays further established Rev7 as a pro-NHEJ factor in budding yeast. Taken together, we report that independent of Shieldin subunits, S. cerevisiae Rev7 functions as an anti-resection and pro-NHEJ factor via direct regulation of the MRX complex. These observations bridge a notable gap in the current understanding of the molecular mechanism underpinning Rev7-mediated regulation of DSB repair pathway choice in S. cerevisiae. Overall, this thesis provides novel insights into the regulation of DNA recombination and repair in S. cerevisiae by two HORMA domain proteins Hop1 and Rev7. These findings present new directions for exploring their functions in regulating these vital processes.