Berardini, Mark Douglas; (1993) In vitro DNA cross-linking and sequence specificity of bifunctional alkylating agents. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

The development and implementation of a simple, sensitive and reliable assay to measure the formation of interstrand cross-links induced in isolated DNA by bifunctional alkylating agents is described. A number of compounds were evaluated for their ability to induce interstrand crosslinks in DNA including the nitrogen mustard class of alkylating agents, several structurally related analogues of the bioreductive aziridinylbenzoquinone AZQ (diaziquone), and several C8-linked pyrrolobenzodiazepine dimers. The assay involves the reaction of end- labeled linear plasmid DNA with cross-linking agents and the subsequent separation and detection of cross-linked DNA from uncross-linked DNA using agarose gel electrophoreses. The technique compares the extent and rate of cross-link formation of different classes of bifunctional agents and can be extended to measure the conversion of DNA monoadducts to crosslinks. In the second part of this study, the nucleotide sequence preferences for the formation of interstrand cross-links induced in DNA by nitrogen mustard, 2,5-diaziridinyl-l,4-benzoquinone (DZQ) and 3,6-dimethyl DZQ (MeDZQ) were examined using synthetic duplex oligonucleotides and denaturing polyacrylamide gel electrophoresis (PAGE). Nitrogen mustard was found to preferentially cross-link DNA in a 5'-GNC sequence using a panel of oligonucleotides containing a single potential cross-linking site. Reaction of DZQ and MeDZQ with DNA duplexes containing several potential cross-linking sites resulted in the formation of cross-linked DNAs with different electrophoretic mobilities. Analysis of the principal cross-linked products by piperidine fragmentation revealed that the preferential site of cross-linking was altered from a 5'-GNC to a 5'-GC sequence upon reduction of DZQ to the hydroquinone form by the enzyme DT-Diaphorase. In contrast, the reduced form of MeDZQ was found to preferentially cross-link at 5'GNC sites within the same sequence. Additional minor cross-linked products were characterised and revealed that unreduced DZQ and reduced MeDZQ are both capable of cross-linking across four base pairs in a 5'-GNNC sequence.

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