The role of recombinational repair in the mechanism of killing during thymine starvation in Escherichia coli

Thymineless death;recombinational repair;thymine starvation;Hydroxyurea;RecA;double strand breaks;chromosomal fragmentation;replication fork breakage;epistatic analysis;pulse field gel electrophoresis

Kuong, Ka Wai

关键词: Thymineless death;    recombinational repair;    thymine starvation;    Hydroxyurea;    RecA;    double strand breaks;    chromosomal fragmentation;    replication fork breakage;    epistatic analysis;    pulse field gel electrophoresis;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/29515/Kuong_KaWai.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Thymineless death (TLD) describes a universal phenomenon in which cells cannot recover after starvation for one of the four DNA precursors, dTTP. The mechanism of how cells lose viability during thymine starvation has remained a mystery for decades. In Escherichia coli, thymine-starved cells were observed to accumulate chromosomal breaks and induce the SOS response, indicating chromosomal damages, but it is unclear if the chromosomal damage is the cause of lethality. The goal of this work is to elucidate the mechanism of thymineless death, by investigating if the killing is caused by formation of irreparable chromosomal lesions, the mechanism of lesion formation, and characterization of the role of recombinational repair in preventing, repairing or generating these chromosomal lesions.I first characterized the extent of DNA replication, stability and damage during TLD using physical methods, such as pulse-field gel electrophoresis to measure levels of double strand breaks. I next investigated the role of major DNA repair and recombinational repair pathways by studying the effect of their inactivation on survival during thymine starvation. Genetic epistatic analysis was done to find out which recombinational repair pathways were beneficial and which ones were detrimental during thymine starvation, as well as what were the main players in each pathway. I checked how various beneficial or detrimental recombinational repair proteins contribute to the chromosomal abnormalities during TLD. I also compared the survival and chromosomal abnormalities of thymine starvation to other cases of abnormal replication, such as in hydroxyurea-treated cells, which are starved for all four DNA precursors but displayed a static rather than lethal effect. While doing so, I discovered the instability of hydroxyurea in solution and identified the decomposition products.In this work, I established the roles of all major recombinational repair proteins, including RecA, RecBCD, RecFOR, RecG, RuvABC and UvrD in thymineless death. I showed that stalling replication forks during thymine starvation leads to chromosomal fragmentation, partly due to replication fork disintegration.I then proposed how parallel futile repair of these chromosomal breaks leads to genotoxicity.A model of how recombinational repair proteins contribute to destruction of replication origin via futile repair of double strand breaks from disintegrated replication forks is proposed at the end.