Alternatives to the DNA precursor-synthesizing enzyme complex hypothesis Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/vq27zr367

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  • Investigators have been studying an aggregate containing enzymes of deoxyribonucleoside triphosphate biosynthesis in T4-phage infected bacteria. They suggest that it behaves as an organized complex, efficiently channeling DNA precursors to the replication apparatus. Previous work has suggested that about 10 enzyme activities remain associated through several fractionation steps. This multi-enzyme aggregate has been reported to sediment through sucrose gradients at 15-20 S. Also, individual activities are kinetically coupled in crude preparations of this aggregate, such that in vitro it can initiate catalysis of multistep pathways with virtually no lag and with low accumulation of intermediates. However, work presented here shows that aggregation of thymidylate synthase, the key enzyme of all kinetic coupling studies, is not observed when either of its two enzyme cofactors is present. Evidence suggests that the enzyme binds to ribosomal subunits due to an affinity for single-stranded nucleic acids under conditions of low ionic strength. This unexpected affinity, present even in the absence of other T4 proteins, can be explained as a consequence of the enzyme being part of the morphological structure of the virus particle. Nucleoside diphosphate kinase and adenylate kinase, two host enzymes of the proposed complex, are shown to sediment with the exact same pattern in uninfected or infected cells. Thus, aggregation of these dNTP-synthesizing enzymes is not influenced qualitatively or quantitatively by the increased number of DNA replication forks present in an infected cell. Contrary to previous reports, sedimentation values in sucrose gradients are 30-50 S irrespective of the number of enzyme activities present in the aggregate. This aggregation is sensitive to physiological concentrations of salt. Evidence suggests that aggregation is due to low-salt induced binding of proteins to ribosomal subunits. In situ evidence of DNA-precursor channeling in sucrose plasmolyzed, 14 infected cells is shown not to be consistent with results obtained with other plasmolysis or gentle lysate systems. The discrepency can be resolved by the demonstration that sucrose plasmolyzed, T4 infected, cells may have relatively intact membranes.
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