Chaudhry M A, Weinfeld M
Radiobiology Program, Cross Cancer Institute, Edmonton, AB, Canada.
J Mol Biol. 1995 Jun 23;249(5):914-22. doi: 10.1006/jmbi.1995.0348.
Energy deposition by ionizing radiation can lead to the formation of clustered DNA damage, i.e. more than one lesion situated within a helical turn of DNA. Among the postulated lesions are those characterized by damaged bases and abasic sites on opposite strands. Enzymatic removal of such lesions may inadvertently lead to the formation of double-strand breaks. To test this hypothesis, we have constructed model substrates containing damaged bases (5,6-dihydrothymine) or abasic sites set one, three, five and seven bases apart on opposite strands, and examined the reactivity of Escherichia coli endonuclease III towards these substrates. Endonuclease III demonstrates two activities; as a glycosylase that removes saturated pyrimidine bases, such as dihydrothymine, and as an AP lyase that cleaves DNA strands at abasic sites. Analysis of endonuclease III-treated dihydrothymidine containing plasmid DNA by agarose gel electrophoresis indicated that the enzyme generated only single-strand breaks when the base damage was set one and three base-pairs apart, and only slowly introduced double-strand breaks in the other substrates. Endonuclease III treatment of the abasic site-containing DNA, however, readily yielded double-strand breaks. Taken together, these results indicate that the glycosylase activity of the enzyme, but not the AP lyase activity, is inhibited by the presence of a closely positioned break in the opposite strand.
电离辐射引起的能量沉积可导致簇状DNA损伤的形成，即DNA螺旋一圈内存在一个以上的损伤。推测的损伤类型包括双链上碱基受损和无碱基位点的情况。酶促去除此类损伤可能会意外导致双链断裂的形成。为了验证这一假设，我们构建了模型底物，这些底物在双链上含有受损碱基（5,6 - 二氢胸腺嘧啶）或无碱基位点，且间隔分别为1、3、5和7个碱基，并检测了大肠杆菌内切核酸酶III对这些底物的反应活性。内切核酸酶III具有两种活性：作为一种糖苷酶可去除饱和嘧啶碱基，如二氢胸腺嘧啶；作为一种脱嘌呤嘧啶内切酶可在无碱基位点切割DNA链。通过琼脂糖凝胶电泳分析内切核酸酶III处理的含二氢胸腺嘧啶的质粒DNA表明，当碱基损伤相隔1和3个碱基对时，该酶仅产生单链断裂，而在其他底物中双链断裂的产生较为缓慢。然而，内切核酸酶III处理含无碱基位点的DNA时，很容易产生双链断裂。综上所述，这些结果表明，该酶的糖苷酶活性，而非脱嘌呤嘧啶内切酶活性，会受到双链上相邻断裂的抑制。
