Sequence-selective interaction of the minor-groove interstrand cross-linking agent SJG-136 with naked and cellular DNA: footprinting and enzyme inhibition studies.
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 BglII, which has a 5'-A-GATC-T-3' motif at its cleavage site.
Martin
Chris
C
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
Ellis
Tom
T
McGurk
Claire J
CJ
Jenkins
Terence C
TC
Hartley
John A
JA
Waring
Michael J
MJ
Thurston
David E
DE
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001570:Benzodiazepinones / Q000378:metabolism* / Q000494:pharmacology; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism* / Q000494:pharmacology; D018983:DNA Footprinting; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000037:antagonists & inhibitors / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods; D011758:Pyrroles / Q000378:metabolism* / Q000494:pharmacology; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013696:Temperature; D014158:Transcription, Genetic
C423343:1,1'-((propane-1,3-diyl)dioxy)bis(7-methoxy-2-methylidene-1,2,3,10,11,11a-hexahydro-5H-pyrrolo(2,1-c)(1,4)benzodiazepin-5,11-dione); D001426:Bacterial Proteins; D001570:Benzodiazepinones; D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004791:Enzyme Inhibitors; D011758:Pyrroles; D016601:RNA-Binding Proteins; C035041:endodeoxyribonuclease MluI; D003850:Deoxyribonuclease I; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific