In vitro characterization of meiotic recombination with a special focus on Mer3 helicase

Abstract

Sexually reproducing organisms produce haploid gametes to pass their genetic information to other organisms as a means of producing offspring. To produce gametes, organisms developed a special type of cell division - meiosis. Meiotic cell division not only reduces the number of chromosomes by half, but also creates new allele combinations via segregation of homologous chromosomes as well as chromosome recombination. Homologous recombination must be carefully regulated to assure accurate chromosome segregation. This includes the formation of connections between the chromosomes as a part of the crossover formation process. Crossovers arise as a result of the double-strand break repair and are only one of the possible break repair outcomes. Although it is not precisely known how the decision is made, we know that some proteins promote and some prevent crossover formation. In my project, I focused on one of the crossover formation promoting proteins - the S. cerevisiae helicase Mer3 (HFM1 in mammals). Mer3 helicase is a member of the ZMM group of proteins that facilitates the formation of class I crossovers during meiosis. I studied the biochemical and structural characteristics of this protein and its interaction with other proteins participating in meiotic recombination. Using multiple methodological approaches, I characterized in more detail the previously described interaction with the Mlh1/Mlh2 complex. I also studied the newly discovered interaction with the recombination-regulating factors Top3 and Rmi1. Top3 and Rmi1 are a part of the Sgs1/Top3/Rmi1 complex, which acts to untangle a variety of other DNA recombination intermediates and acts against crossover formation. The results of my studies are summarized in Chapter 2, “Integrated structural study of the Mer3 helicase reveals a novel role in promoting meiotic crossovers”, which is also a manuscript prepared for submission. In Chapter3, I explore the current knowledge about meiotic helicases, their interaction partners, and the role of regulatory modifications during meiosis I. I also focus on the molecular structure and mechanisms of these helicases. The fourth chapter includes the manuscript published in 2020 – “Biochemical and functional characterization of a meiosis-specific Pch2/ORC AAA+ assembly”. The paper includes my in vitro analysis, which provided biochemical insights into our knowledge about the interaction between Pch2 and the ORC complex. During my doctorate, I also aimed to solve the Mer3 structure using different experimental techniques: crystallization and CryoEM. Although these experiments did not result in solving the structure of Mer3, I include this part of my project in my thesis together with other important preliminary data. The outcome of these experiments, due to their potential, may be the basis for future research projects. Taken together, my results lead to a better understanding of the roles of the proteins involved in the regulation of meiotic cell division and will be a solid base for future work that can reveal the complete chain of events that happen during the meiotic division.