Sequence-Selective Interaction of the Minor-Groove Interstrand Cross-Linking Agent SJG-136 with Naked and Cellular DNA:  Footprinting and Enzyme Inhibition Studies†

Martin, C., Ellis, T., McGurk, C.J., Jenkins, T.C., Hartley, J.A., Waring, M.J. and Thurston, D.E. (2005) Sequence-Selective Interaction of the Minor-Groove Interstrand Cross-Linking Agent SJG-136 with Naked and Cellular DNA:  Footprinting and Enzyme Inhibition Studies†. Biochemistry, 44 (11). pp. 4135-4147. /10.1021/bi0479813.

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DOI: /10.1021/bi0479813

Abstract

SJG-136 (3) is a novel pyrrolobenzodiazepine (PBD) dimer that is predicted from molecular models to bind in the minor groove of DNA and to form sequence-selective interstrand cross-links at 5′-Pu-GATC-Py-3′ (Pu = purine; Py = pyrimidine) sites through covalent bonding between each PBD unit and guanines on opposing strands. Footprinting studies have confirmed that high-affinity adducts do form at 5′-G-GATC-C-3′ sequences and that these can inhibit RNA polymerase in a sequence-selective manner. At higher concentrations of SJG-136, bands that migrate more slowly than one of the 5′-G-GATC-C-3′ footprint sites show significantly reduced intensity, concomitant with the appearance of higher molecular weight material near the gel origin. This phenomenon is attributed to interstrand cross-linking at the 5′-G-GATC-C-3′ site and is the first report of DNA footprinting being used to detect interstrand cross-linked adducts. The control dimer GD113 (4), of similar structure to SJG-136 but unable to cross-link DNA due to its C7/C7′-linkage rather than C8/C8′-linkage, neither produces footprints with the same DNA sequence nor blocks transcription at comparable concentrations. In addition to the two high-affinity 5′-G-GATC-C-3′ footprints on the MS2 DNA sequence, other SJG-136 adducts of lower affinity are observed that can still block transcription but with lower efficiency. All these sites contain the 5′-GXXC-3′ motif (where XX includes AG, TA, GC, CT, TT, GG, and TC) and represent less-favored cross-link sites. In time-course experiments, SJG-136 blocks transcription if incubated with a double-stranded DNA template before the transcription components are added; addition after transcription is initiated fails to elicit blockage. Single-strand ligation PCR studies on a sequence from the c-jun gene show that SJG-136 binds to 5′-GAAC-3′/5′-GTTC-3′ (preferred) or 5′-GAGC-3′/5′-GCTC-3′ sequences. Significantly, adducts are obtained at the same sequences following extraction of DNA from drug-treated K562 cells, confirming that the agent reaches the cellular genome and interacts with the DNA in a sequence-selective fashion. Finally, SJG-136 efficiently inhibits the action of restriction endonuclease Bg/IL, which has a 5′-A-GATC-T-3′ motif at its cleavage site.

Item Type:Article
Departments, units and centres:Department of Pharmaceutical and Biological Chemistry > Department of Pharmaceutical and Biological Chemistry
ID Code:2909
Journal or Publication Title:Biochemistry
Deposited By:Library Staff
Deposited On:08 Mar 2012 10:52
Last Modified:08 Mar 2012 10:52

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