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

详细信息    查看全文

• 作者：Chris Martin ; Tom Ellis ; Claire J. McGurk ; Terence C. Jenkins ; John A. Hartley ; Michael J. Waring ; and David E. Thurston
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：March 22, 2005
• 年：2005
• 卷：44
• 期：11
• 页码：4135 - 4147
• 全文大小：774K
• 年卷期：v.44,no.11(March 22, 2005)
• ISSN：1520-4995

文摘

SJG-136 (3) is a novel pyrrolobenzodiazepine (PBD) dimer that is predicted from molecular models to bindin 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. Footprintingstudies have confirmed that high-affinity adducts do form at 5'-G-GATC-C-3' sequences and that these can inhibit RNApolymerase in a sequence-selective manner. At higher concentrations of SJG-136, bands that migrate more slowly than oneof the 5'-G-GATC-C-3' footprint sites show significantly reduced intensity, concomitant with the appearance of highermolecular 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. Thecontrol dimer GD113 (4), of similar structure to SJG-136 but unable to cross-link DNA due to its C7/C7'-linkage ratherthan C8/C8'-linkage, neither produces footprints with the same DNA sequence nor blocks transcription at comparableconcentrations. In addition to the two high-affinity 5'-G-GATC-C-3' footprints on the MS2 DNA sequence, other SJG-136adducts of lower affinity are observed that can still block transcription but with lower efficiency. All these sites contain the5'-GXXC-3' motif (where XX includes AG, TA, GC, CT, TT, GG, and TC) and represent less-favored cross-link sites. Intime-course experiments, SJG-136 blocks transcription if incubated with a double-stranded DNA template before thetranscription components are added; addition after transcription is initiated fails to elicit blockage. Single-strand ligationPCR 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 DNAfrom drug-treated K562 cells, confirming that the agent reaches the cellular genome and interacts with the DNA in asequence-selective fashion. Finally, SJG-136 efficiently inhibits the action of restriction endonuclease BglII, which has a5'-A-GATC-T-3' motif at its cleavage site.