Cells deficient in the base excision repair protein, DNA polymerase beta, are hypersensitive to oxaliplatin chemotherapy

A significant proportion of human cancers overexpress DNA polymerase beta (Pol beta), the major DNA polymerase involved in base excision repair. The underlying mechanism and biological consequences of overexpression of this protein are unknown. We examined whether Pol beta, expressed at levels found in tumor cells, is involved in the repair of DNA damage induced by oxaliplatin treatment and whether the expression status of this protein alters the sensitivity of cells to oxaliplatin. DNA damage induced by oxaliplatin treatment of HCT116 and HT29 colon cancer cells was observed to be associated with the stabilization of Pol beta protein on chromatin. In comparison with HCT116 colon cancer cells, isogenic oxaliplatin-resistant (HCT-OR) cells were found to have higher constitutive levels of Pol beta protein, faster in vitro repair of a DNA substrate containing a single nucleotide gap and faster repair of 1,2-GG oxaliplatin adduct levels in cells. In HCT-OR cells, small interfering RNA knockdown of Pol beta delayed the repair of oxaliplatin-induced DNA damage. In a different model system, Pol beta-deficient fibroblasts were less able to repair 1,2-GG oxaliplatin adducts and were hypersensitive to oxaliplatin treatment compared with isogenic Pol beta-expressing cells. Consistent with previous studies, Pol beta-deficient mouse fibroblasts were not hypersensitive to cisplatin treatment. These data provide the first link between oxaliplatin sensitivity and DNA repair involving Pol beta. They demonstrate that Pol beta modulates the sensitivity of cells to oxaliplatin treatment. Oncogene (2010) 29, 463-468; doi:10.1038/onc.2009.327; published online 19 October 2009

Detection of DNA base variation and cytosine methylation at a single nucleotide site using a highly sensitive fluorescent probe

A fluorescent DNA probe containing an anthracene group attached via an anucleosidic linker can identify all four DNA bases at a single site as well as the epigenetic modification C/5-MeC via a hybridisation sensing assay.

Anthracene-modified oligonucleotides as fluorescent DNA mismatch sensors: discrimination between various base-pair mismatches

A fluorescent anthracene-tagged DNA probe has been shown to respond to various DNA sequences by changes to its emission signal upon duplex formation. The fluorescence response for duplexes containing a single mismatch near the anthracene site has been found to be very sensitive to its composition, with the emission signal increasing for a CA mismatch and decreasing for CT and CC mismatches.

Label-Free Detection of Single-Base Mismatches in DNA by Surface-Enhanced Raman Spectroscopy

Singles only: DNA sequences can be induced to spontaneously adsorb to the surfaces of Ag colloids through their nucleotide side chains (see picture). The SERS spectra of these nonspecifically bound strands are sufficiently reproducible that they can be used to identify single-base mismatches in short (25-mer and 23-mer) strands. Subtracting the spectra of different DNA sequences results in difference spectra that contain features corresponding to the exchanged nucleotides.

RESONANCE RAMAN-SPECTRA OF THE TRIPLET-STATE OF FREE-BASE TETRAPHENYLPORPHYRIN AND 6 OF ITS ISOTOPOMERS

The resonance Raman spectra of the ground state and the lowest excited tripler state of free-base tetraphenylporphyrin and six of its isotopomers have been obtained using two-color time-resolved techniques. Ground-state spectra were recorded using low-energy 447 nm probe laser pulses, and triplet-state spectra were probed, with similar pulses, 30 ns after high-energy excitation with 532 nm pump pulses. Polarization data on both the ground and triplet states are also reported. The resonance Raman spectrum of the triplet is very different from that of the ground state but the combination of extensive isotope substitution with polarization data allows bands in the ground state to be assigned and corresponding bands in the tripler state to be located. Isotope shifts of the same bands in the S-0 and T-1 states are similar, implying that the compositions of the vibrational modes do not change significantly on excitation. Two of the strongest bands in the T-1 spectra are associated with phenyl ring substituents; these are shifted less than 5 cm(-1) between the S-0 and T-1 states so that bonding in the phenyl substituents is barely affected by excitation to the T-1 state. The changes in position of the porphyrin ring bands are larger, but still only tens of cm(-1) or less, the main changes in the spectra being due to differences in relative band intensities in the two states. The relatively small shifts in the porphyrin ring band positions which are observed show that the excitation energy is not localized on a single small region of the molecule but is delocalized over the entire porphyrin skeleton. This picture of an excited species with high chemical reactivity, but with individual bonds only slightly perturbed from the ground state, is contrasted with molecules, such as benzophenone, where excitation causes a large perturbation in the bonding within a single functional group.

TIME-RESOLVED RESONANCE RAMAN-SPECTROSCOPY OF TRIPLET-STATE METALATED AND FREE-BASE TETRAARYLPORPHYRINS

Resonance Raman spectra of the T-1 excited states of Zn and free-base tetra-4-sulfonatophenylporphyrin (TPPS) have been recorded at room temperature in aqueous solution using two-colour time-resolved methods. The spectra of both sulfonated molecules are very similar to their tetraphenylporphyrin (TPP) analogues, which have been recorded in THF solution using the same pump-probe conditions, but they have higher signal-to-noise ratios because interference from strong solvent bands is reduced. Although two different T-1 spectra of Zn(TPP) have been reported these spectra differ slightly from each other and from the spectrum reported here, which has band positions very close (+/-6 cm(-1)) to those of Zn(TPPS). The high S/N ratios obtainable for the water-soluble porphyrins have allowed reliable polarization data to be recorded for their S-0 and T-1 states. This data set allows a realistic comparison of the changes in bonding associated with excitation of both free-base and Zn tetraarylporphyrins to the T-1 state.

TIME-RESOLVED RESONANCE RAMAN-SPECTROSCOPY OF EXCITED SINGLET AND TRIPLET-STATES OF FREE-BASE MESO-TETRAPHENYLPORPHYRIN

Two-color time-resolved resonance Raman spectroscopy has been used to probe the lowest excited singlet (S1) and triplet (T1) states of free-base meso-tetraphenylporphyrin and meso-tetrakis(4-sulphonatophenyl)porphyrin in solution at room temperature. The spectra were recorded using 532-nm excitation pulses and time-delayed probe pulses (DELTAT = 0-30 ns, 447 and 460 nm) near lambda(max) of the S1 and T1 states. Significant shifts in frequency of the porphyrin core vibrations were observed upon excitation to either the S1 or T1 state. Several of the strongest polarized bands in the spectra of both excited states, including nu1, nu2, nu4, nu6, and phi4, are assigned, and the information they give on the differences in electron distribution in the ground, S1, and T1 states is discussed.