MONOFUNCTIONAL ANGULAR FUROCOUMARINS: SEQUENCE SPECIFICITY IN DNA HOTOBINDING OF 6,4,4′‐TRIMETHYLANGELICIN and OTHER ANGELICINS

Abstract

The sequence specificity in the photoreaction (365 nm) of 6,4,4'-trimethylangelicin (TMA) with DNA fragments of the lac I gene of Escherichia coli was studied by using DNA sequencing methodology. In order to map the sites of TMA photoaddition, we took advantage of the (3'-5') exonuclease activity associated with T4 DNA polymerase, which is blocked by bulky adducts, such as furocoumarin photoadducts. A quantitative analysis of the sites of photoaddition is reported. TMA was demonstrated to photoreact with thymine and, to a lower extent, to cytosine. AT-rich sequences and TTT sites in a GC context are the most reactive sites towards TMA whereas TA, AT, CA, AC sites are weaker sites with similar reactivity. Cytosines in alternated CG sequences are also targets of TMA photobinding. We observed a less pronounced sequence specificity of TMA than that of other psoralen derivatives already studied (Sage and Moustacchi, 1987; Boyer et al., 1988). A comparison with other furocoumarins 4,4'-dimethylangelicin (4,4'-DMA), 4'-methylangelicin (4'-MA), angelicin, 4,5',8-trimethylpsoralen (TMP) and 8-methoxypsoralen (8-MOP) is also reported. The role of flanking sequence and consequently of the local conformation at the various sites of photoaddition is discussed. A preferential orientation of the TMA molecule during the intercalation in the dark is suggested. Hot alkali treatment of TMA-modified DNA did not reveal any DNA strand breakage due to photooxidized bases.

Strand Specificity of Mutagenic Bypass Replication of DNA Containing Psoralen Monoadducts in a Human Cell Extract

“…Adducts have been reported at cytidines in 5Ј-CpA and 5Ј-ApC contexts, yet modification of 5Ј-CpG has not been observed (21). Hence, the PUVA-dependent increases in error rates for T ⅐ A3C ⅐ G, C ⅐ G3G ⅐ C, and C ⅐ G3A ⅐ T events (Table 2) imply that most of these result from targeted error-prone replication of MAs.…”

Recent Advances in Psoralen Phototoxicity Mechanism

“…In comparison to other psoralen derivatives, TMA showed a lower sequence specificity than 8-MOP possibly due to its high affinity to DNA, to its angular structure and to the presence of the methyl groups. Unlike 8-MOP , there were no hot-spots for TMA photoaddition (Miolo et al, 1989a). The base specificity of 4,4',6-TMA to a single-stranded DNA was also determined (Miolo et a/.…”

Rapid characterization of cross-links, mono-adducts, and non-covalent binding of psoralens to deoxyoligonucleotides by LC-UV/ESI-MS and IRMPD mass spectrometry

“…18 Several studies agree that the 5 0 -TA site is more reactive than the 5 0 -AT site, 11,15,16,[19][20][21][22][23][24] and that repeated T-A sequences are hot spots for reaction due to the increased number of potential binding sites. 15,[24][25][26] Determination of sequence selectivities and characterization of the resulting adducts and/or cross-linked products remains a significant analytical challenge, both for psoralens and for other classes of DNA interactive agents that engage in covalent binding to DNA.…”