Pierce J R, Nazimiec M, Tang M S
M. D. Anderson Cancer Center, University of Texas, Smithville 78957.
Biochemistry. 1993 Jul 20;32(28):7069-78. doi: 10.1021/bi00079a002.
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-.
通过核酸外切酶III（exo III）消化、λ核酸外切酶（λ exo）消化和UvrABC核酸酶切口分析，确定了两种吡咯并[1,4]苯并二氮杂卓衍生物——托马霉素（Tma）和氨茴霉素（Atm）——的DNA结合位点。exo III消化在药物-DNA加合物的3'端4-5个碱基处停滞。虽然这种方法可以识别大多数Atm和Tma-DNA修饰位点，但它很复杂，因为exo III消化也会被某些未修饰的序列以及与相反链结合的药物所停滞。λ exo消化在药物-DNA加合物的5'端1-2个碱基处停滞。λ exo方法也能识别大多数药物-DNA结合位点，并产生更清晰的背景；然而，它也受到相反链药物结合的影响。由于它们固有的消化极性，这两种核酸外切酶往往会被DNA分子一端的药物-DNA加合物所停滞。纯化的UvrA、UvrB和UvrC蛋白共同作用，在Atm或Tma-DNA加合物的5'端6-8个碱基和3'端4个碱基处进行双重切口。这种核酸酶复合物识别通过核酸外切酶消化方法确定的所有Tma和Atm-DNA结合位点，并且所有UvrABC切口都可归因于被切割DNA链中的药物修饰。UvrABC核酸酶切口的程度随着DNA修饰药物浓度的增加而增加。使用UvrABC切口方法，我们确定了Tma和Atm-DNA加合物在三个DNA片段中形成的序列偏好，并且我们发现这两种药物具有不同的加合偏好位点。Tma和Atm-DNA结合都受到5'和3'相邻碱基的强烈影响；Tma的5'和3'优先碱基顺序是A > G，T > C，以及A，C > G，T，而Atm的顺序是A > G > T > C和A > G > T，C。Tma结合的优先三联体是-AGA- > -GGC-，-TGC-，和AGC-，而Atm的是-AGA-，-AGG- > -GGA-，-GGG-。
