Cancer and bacterial infections are caused by uncontrolled multiplication of undesired cell types within the organism. Effective treatments target critical and specific parts of these undesired cells, absent in by-stander cells of the organism. One successful strategy to fight cancer or infections is to target the complex DNA metabolism of the undesired cells, causing their chromosomal death, ? a mysterious phenomenon, whose mechanisms are still completely unclear. Chromosomal death is the inability to continue chromosome cycle ?the cycle of replication and segregation of genetic information that drives the cell cycle. The chromosomal cycle is blocked by chromosomal lesions ? DNA lesions that are so complex and harmful, that they inactivate the entire chromosomes. A classic chromosomal lesion is a double-strand DNA break, but the most common chromosomal lesions are efficiently mended by pathways of recombinational repair and various back-ups. We are purposefully looking for and characterizing conditions that induce irreparable chromosomal lesions that cause chromosomal death, to make it possible to convert them into future treatments against undesirable cells. This application describes our characterization of two such conditions: (i) thymine starvation; (ii) toxic RNA incorporation into DNA. In characterizing a chromosome- associated lethal phenomenon, we practice a three-pronged approach. On the one hand, we identify the most dramatic physical readouts; on the other hand, we identify the most interesting phenotypes of mutants. The third arm of the analysis is to look at the chromosome from the genome perspective. Our fist specific aim will address genetic and metabolic aspects of thymine starvation in relation to its three distinct phases. The second specific aim will characterize physical changes in the chromosome during thymine starvation.
The third aim i s about comprehensive characterization of the novel phenomenon of the unexpected RNA nucleotide toxicity within the chromosomal DNA. Combining the most relevant physical readouts with the most interesting mutants and the most dramatic genomic patterns should yield a comprehensive 3D-picture of the phenomenon in genetic / physical / genomic dimensions, producing mechanistic insights into the most complicated mechanisms of chromosomal death.
Compared to exogenous DNA damage, which usefulness is limited by toxicity to by-stander cells, cell-type-specific induction of endogenous DNA damage is a much safer approach to effect chromosomal death in undesirable cell types without affecting by-standers. This research program investigates how inhibition of two specific segments of the DNA replication metabolism causes chromosomal death in bacteria. The obtained information will lay a foundation for future design of highly-specific and potent anti-cancer and anti-infection treatments.
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