Comparison of sequence preference of tomaymycin- and anthramycin-DNA bonding by exonuclease III and .lambda. exonuclease digestion and UvrABC nuclease incision analysis

Abstract

The DNA bonding sites of two pyrrolo[1,4]benzodiazepine derivatives--tomaymycin (Tma) and anthramycin (Atm)--were identified by exonuclease III (exo III) digestion, lambda exonuclease (lambda exo) digestion, and UvrABC nuclease incision analysis. exo III digestion stalls 4-5 bases 3' to a drug-DNA adduct. While this method can recognize most of the Atm-and Tma-DNA modification sites, it is complicated in that exo III digestion is also stalled by certain unmodified sequences and by drug bound to the opposite strand. lambda exo digestion stalls 1-2 bases 5' to a drug-DNA adduct. The lambda exo method also recognizes most of the drug-DNA bonding sites and renders a cleaner background; however, it is also affected by opposite-strand drug bonding. Due to their intrinsic digestion polarities, these two exonucleases tend to be stalled by the drug-DNA adduct at one end of the DNA molecule. Purified UvrA, UvrB, and UvrC proteins acting together make dual incisions 6-8 bases 5' and 4 bases 3' to a Atm- or Tma-DNA adduct. This nuclease complex recognizes all the Tma- and Atm-DNA bonding sites identified by exonuclease digestion methods, and all the UvrABC incisions can be attributed to drug modifications in the incised DNA strand. The degree of UvrABC nuclease incision increases with increasing drug concentrations for DNA modification. Using the UvrABC incision method, we have identified the sequence preference of Tma- and Atm-DNA adduct formation in three DNA fragments, and we have found that these two drugs have different preferred sites for adduction. Both Tma- and Atm-DNA bonding is strongly influenced by the 5' and 3' neighboring bases; the orders of preferred 5' and 3' bases for Tma are A > G, T > C, and A, C > G, T, and for Atm the orders are A > G > T > C and A > G > T, C. The preferred triplets for Tma bonding are -AGA- > -GGC-, -TGC-, and AGC- and for Atm are -AGA-, -AGG- > -GGA-, -GGG-.

Human Transcription Release Factor 2 Dissociates RNA Polymerases I and II Stalled at a Cyclobutane Thymine Dimer

“…5B. Digestion with T4 DNA polymerase 3Ј to 5Ј exonuclease in the absence of transcription generates a fragment of 50 nt, consistent with a previous report of block of T4 polymerase 3Ј to 5Ј exonuclease immediately at the dimer (30). When RNA polymerase I is present under transcription conditions, in addition to the 50-mer arising from the nontranscribed DNA, two specific bands of 73 and 74 nt are observed (Fig.…”

“…There is extensive literature on transcription-coupled repair of genes transcribed by RNA polymerase II (1)(2)(3)(4)(5)30). In contrast, there are only a few studies addressing the questions of the effects of lesions in the rRNA genes on RNA polymerase I and the effect of RNA polymerase I transcription on repair of rRNA genes (38 -43).…”

PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti‐tumour activity

“…These preferences are different from those of other DNA minor groove binders that alkylate DNA. For example, anthramycin selects AGA and AGG triplets (Pierce et al ., 1993), saframycins prefer GGG or GGC (Rao and Lown, 1992) while CC‐1065 and derivatives (adozelesin, bizelesin), as well as duocarmycin, prefer the minor groove of AT‐rich regions (Baraldi et al ., 1999). On the other hand, preferential triplets for PM01183 binding were similar to those previously reported for trabectedin (TGG, CGG, AGC and GGC) (Marco et al ., 2006) and Zalypsis ® (GGC and AGC) (Leal et al ., 2009).…”

Sensitive Detection of Small-Molecule Targets Using Cooperative Binding Split Aptamers and Enzyme-Assisted Target Recycling

“…25 Additionally, target binding within the aptamer can interfere with enzyme binding and activity; for example, some small-molecule DNA-intercalating agents and DNA adducts can inhibit nucleases, especially exonucleases. 24,26,27 …”