The broad goal of this study is to investigate the chromosomal consequences of cell's inability to maintain the three critical aspects of the DNA precursors pools: their quantity, their balance and their purity. Quality of DNA precursor pools has been long recognized for its important role in avoidance of mutagenesis, which is significant for evolution of any species. Recently, we have argued for a more immediate consequence of limited, imbalanced or contaminated DNA precursor pools, which is formation of chromosomal lesions that threaten cell's survival and that require complex DNA mending system called recombinational repair. We have proposed two general models of how base analog incorporation into DNA could lead to chromosomal fragmentation that requires recombinational repair. In our previous work, we identified uracil and hypoxanthine as the major base analogs contaminating DNA precursor pools in E. coli. At the same time, the identity and the sources of other natural base analogs contaminating DNA precursor pools remain unknown. Moreover, even though DNA precursor pool imbalance at first leads to reparable chromosomal lesions, it eventually generates irreparable chromosomal lesions of unknown nature. Our recent observations provide insights into the possible scenarios leading to irreparable chromosomal lesions and into the nature of these lesions, while introduction of genome analysis presents an opportunity to identify the most affected parts of the chromosome.
Aim 1 of this study addresses specific mechanisms of chromosomal fragmentation, induced by hypoxanthine, uracil or fluorouracil.
Aim 2 focuses on characterization of the mysterious origin DNA disappearance during thymine starvation, coinciding with thymineless death.
Aim 3 tests our recently-proposed multi-stage model for thymineless death, which begins with stalling of the existing replication forks, proceeds through unknown number of intermediate stages and ends with the replication origin destruction. Collectively, this work will emphasize the importance to keep DNA precursor pools plentiful, balanced and sanitized by characterizing various sources of imbalance and contamination of the pools, as well as the chromosomal consequences, including irreparable lesions, that result from pool contamination and imbalance.
The bactericidal power of manipulations with the DNA precursor pools is a basis of effective anti-microbial and anti-cancer treatments, with drugs like hydroxyurea, trimethoprim or fluorouracil being standard components of the treatment cocktails, but their mode of action, especially at the level of chromosome, is still unclear. We will characterize the mechanisms of base analog-induced chromosomal fragmentation, the various chromosomal consequences of the DNA precursor pool imbalances, and how they lead to inactivation of the chromosome and genetic death. The results will be broadly relevant to all medically-relevant aspects of DNA metabolism, from folate limitation to designing of better ways to kill undesired cells genetically.
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