Chemical and biological studies of the major DNA adduct of cis-diamminedichloroplatinum(II), cis-(Pt(NH/sub 3/)/sub 2//d(GpG)/), built into a specific site in a viral genome

Journal Article · Tue Jun 14 00:00:00 EDT 1988 · Biochemistry; (United States)

OSTI ID:6895425

Naser, L J; Pinto, A L; Lippard, S J; Essigmann, J M

A duplex Escherichia coli bacteriophage M13 genome was constructed containing a single cis-(Pt(NH/sub 3/)/sub 2//d(GpG)/) intrastrand cross-link, the major DNA adduct of the anticancer drug cis-diamminedichloroplatinum(II). The duplex dodecamer d(AGAAGGCCTAGA) x d(TCTAGGCCTTCT) was ligated into the HincII site of M13mp18 to produce an insertion mutant containing a unique StuI restriction enzyme cleavage site. A genome with a 12-base gap in the minus strand was created by hybridizing HincII-linearized M13mp18 duplex DNA with the single-stranded circular DNA of the 12-base insertion mutant. Characterization by pH-dependent /sup 1/H NMR spectroscopy established that platinum binds to the N7 positions of the adjacent guanosines. The platinated oligonucleotide was phosphorylated in the presence of (..gamma..-/sup 32/P)ATP with bacteriophage T4 polynucleotide kinase and incorporated into the 12-base gap of the heteroduplex, thus situating the adduct specifically within the StuI site in the minus strand of the genome. The site of incorporation of the dodecamer was mapped to the expected 36-base region delimited by the recognition sites of XbaI and HindIII. Gradient denaturing gel electrophoresis of a 289-base-pair fragment encompassing the site of adduction revealed that the presence of the cis-(Pt(NH/sub 3/)/sub 2//d)GpG)/) cross-link induces localized weakening of the DNA double helix. Comparative studies revealed no difference in survival between platinated and unmodified double-stranded genomes. In contrast, survival of the single-stranded platinated genome was only 10-12% that of the corresponding unmodified single-stranded genome, indicating that the solitary cis-(Pt(NH/sub 3/)/sub 2//d(GpG)/) cross-link is lethal to the single-stranded bacteriophage.

• Research Organization: Massachusetts Institute of Technology, Cambridge (USA)
• OSTI ID: 6895425
• Journal Information: Biochemistry; (United States), Vol. 27:12
• Country of Publication: United States
• Language: English

Related Subjects

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
59 BASIC BIOLOGICAL SCIENCES
DNA
CROSS-LINKING
DNA ADDUCTS
MOLECULAR STRUCTURE
PLATINUM COMPOUNDS
ATP
BACTERIOPHAGES
ELECTROPHORESIS
ESCHERICHIA COLI
MUTANTS
PHOSPHORUS 32
ADDUCTS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PARASITES
PHOSPHORUS ISOTOPES
POLYMERIZATION
RADIOISOTOPES
TRANSITION ELEMENT COMPOUNDS
VIRUSES
560300* - Chemicals Metabolism & Toxicology
550201 - Biochemistry- Tracer Techniques