Functions of bacterial type IA topoisomerases in the prevention of R-loop-dependent and -independent genomic instability.

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A key step in the life of a bacterial cell is its division to generate two identical daughter cells. This event allows its perpetuation from generation to generation. However, one key event that must be faithfully accomplish before cell division, is the segregation of the replicated genetic material (chromosomes) to each end of the cell while maintaining the stability of the genome, so that each daughter cell can receive one chromosome following division. With a full set of the genes from the mother cell, each daughter cell will be able to grow and divide. Because of the double-helical nature of the DNA, entanglement of the DNA strands inevitably occurs during replication. If not properly and rapidly removed, these intertwines will eventually block the progression of the replication complex and, as a consequence, DNA damages will occur and chromosome segregation will not be possible. DNA topoisomerases (topos), by cutting DNA and by allowing strand passage and re-ligation solve the topological problem associated with the progression of replication complexes. Type IA topos use ssDNA as substrates, they are the sole ubiquitous topos and many of them possess RNA topo activity. The last two unique features strongly support the hypothesis that type 1A topos were present very early in evolution, i.e., at least in the last universal common ancestor (LUCA) and possibly in the RNA world. Type IA topoisomerases have been shown to play major roles in supercoiling regulation and genome maintenance. Our recent results indicate that type 1A enzymes from the model organism Escherichia coli, maintain the stability of the genome by inhibiting the formation of R-loops, which threaten the stability of the genome by mechanism(s) that are not well characterized. Moreover, we have uncovered a genetic interaction between type IA topos and proteins of the nucleotide excision DNA repair pathway. The main goal of our research over the next five years is to characterize the precise mechanisms by which E. coli type IA topos act on the genome to maintain its stability. To achieve our objective, we will use classical bacterial and molecular genetics as well as state-of-the-art single-cell and genome-wide approaches. The ultimate goal of our research program is to understand how DNA topology affects genome stability and how it is properly regulated by topos, to allow the faithful transmission of the genetic material to daughter cells.