Rifampicin Toxicity and Linear DNA Degradation Dependence of

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					    Rifampicin Toxicity and Linear DNA Degradation
         Dependence of Escherichia coli seqA mutants
                         Tulip Mahaseth and Andrei Kuzminov

       The Escherichia coli SeqA protein negatively regulates initiation of DNA
replication; consequently, seqA mutants exhibit over-initiation and asynchronous
replication phenotypes. Rifampicin, a transcription inhibitor, kills seqA by 3-4 orders of
magnitude in concentrations which are bacteriostatic to wild type cells. In the seqA
mutant upon rifampicin treatment, replication forks can finish only half of ongoing
replication rounds, suggesting that synthesis of certain proteins is required for fork
progression in the absence of SeqA. In order to identify the factors that would make seqA
mutants resistant to rifampicin, I will enrich for multicopy clones of chromosomal DNA
that would allow the seqA mutant to survive rifampicin treatment.
       Moreover, the seqA mutant suffers from increased chromosomal fragmentation,
making the recombinational repair proteins RecBCD and RuvABC, and to a lesser extent
RecA, essential for survival. However, I have found that seqA depends more on the
RecBCD-catalyzed linear DNA degradation than on recombinational repair for its
survival. In order to study how the seqA mutants are saved by linear DNA degradation, I
am going to complement seqA recBCD mutant with plasmids expressing various
RecBCD mutant enzymes and test these transformants’ viability in relation to their
recombinational repair proficiency and linear DNA degradation activity. Furthermore,
this dependence on linear DNA degradation has led us to propose a model which would
explain not only this curious requirement, but also, the seqA mutant’s unique sensitivity
to rifampicin.

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