Effects of monofunctional DNA alkylating agents adozelesin, Et743 and hedamycin on DNA replication and helicase function
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Highly cytotoxic monofunctional DNA alkylators: adozelesin, Et743 and hedamycin, bind double-stranded DNA (dsDNA) and inhibit DNA synthesis. Both adozelesin and Et743 are minor groove binders, however, while adozelesin fits well within, Et743 partially protrudes outside the minor groove and bends it towards the major groove. Hedamycin intercalates the DNA major groove via a threading mechanism, locally unwinds DNA and interacts with both major and minor grooves. Part of this thesis presents findings on replication inhibition by Et743 using simian virus 40 (SV40) as a model system. Analysis of SV40 replication intermediates isolated from virus infected cells, revealed Et743 induced inhibition of SV40 origin activity, which was coincident with accumulation of unusual replication intermediates, indicative of regressed replication forks. Et743 adducts were detected within intracellular SV40 DNA, but at a frequency insufficient to inhibit replication directly on the template. Et743 inhibition of intracellular replication and induction of fork regression likely involves activation of an intra-S-phase checkpoint response. Et743 induced replication inhibition was compared to the effects of the related minor groove DNA alkylators, tomamycin and saframycin A that unlike Et743 do not bend DNA towards the major groove. Interestingly, tomamycin inhibited SV40 origin activity, but unlike Et743, did not cause accumulation of unusual replication intermediates. The second part of this thesis examined the ability of adozelesin, Et743 and hedamycin to block DNA unwinding using as a model enzyme combination helicase/nuclease, RecBCD of Escherichia coli under cell-free conditions. This study characterized anti-helicase properties of adozelesin, Et743 and hedamycin in the context of altered DNA nuclease and ATPase activities of RecBCD. Inhibition of unwinding by DNA alkylating agents was determined using a real time fluorometric assay, which monitors the appearance of single-stranded DNA (ssDNA). Electrophoretic analysis of DNA unwinding products confirmed alkylator-induced inhibition of unwinding and revealed drug-specific effects on RecBCD nucleolytic activities. Inhibition of DNA unwinding induced by adozelesin and Et743 was coincident with increased utilization of ATP by RecBCD. Minor groove binders, adozelesin and Et743 were much more effective inhibitors of DNA unwinding by RecBCD than was the DNA intercalator, hedamycin. Agent specific stress responses were investigated in Bloom helicase (Blm) deficient cells. Analysis of DNA alkylating agent's effects on replication and helicase function could help in defining the role of specific enzymes, such as Blm helicase, during genotoxic stress in cells.