Cannan Wendy J, Tsang Betty P, Wallace Susan S, Pederson David S
From the Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405.
From the Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont 05405
J Biol Chem. 2014 Jul 18;289(29):19881-93. doi: 10.1074/jbc.M114.571588. Epub 2014 Jun 2.
Exposure to ionizing radiation can produce multiple, clustered oxidative lesions in DNA. The near simultaneous excision of nearby lesions in opposing DNA strands by the base excision repair (BER) enzymes can produce double-strand DNA breaks (DSBs). This attempted BER accounts for many of the potentially lethal or mutagenic DSBs that occur in vivo. To assess the impact of nucleosomes on the frequency and pattern of BER-dependent DSB formation, we incubated nucleosomes containing oxidative damages in opposing DNA strands with selected DNA glycosylases and human apurinic/apyrimidinic endonuclease 1. Overall, nucleosomes substantially suppressed DSB formation. However, the degree of suppression varied as a function of (i) the lesion type and DNA glycosylase tested, (ii) local sequence context and the stagger between opposing strand lesions, (iii) the helical orientation of oxidative lesions relative to the underlying histone octamer, and (iv) the distance between the lesion cluster and the nucleosome edge. In some instances the binding of a BER factor to one nucleosomal lesion appeared to facilitate binding to the opposing strand lesion. DSB formation did not invariably lead to nucleosome dissolution, and in some cases, free DNA ends resulting from DSB formation remained associated with the histone octamer. These observations explain how specific structural and dynamic properties of nucleosomes contribute to the suppression of BER-generated DSBs. These studies also suggest that most BER-generated DSBs will occur in linker DNA and in genomic regions associated with elevated rates of nucleosome turnover or remodeling.
暴露于电离辐射可在DNA中产生多个成簇的氧化损伤。碱基切除修复（BER）酶对相对DNA链上附近损伤的近乎同时切除可产生双链DNA断裂（DSB）。这种尝试性的BER解释了体内发生的许多潜在致死性或致突变性DSB的产生原因。为了评估核小体对BER依赖性DSB形成频率和模式的影响，我们将含有相对DNA链上氧化损伤的核小体与选定的DNA糖基化酶和人脱嘌呤/脱嘧啶内切核酸酶1一起孵育。总体而言，核小体显著抑制了DSB的形成。然而，抑制程度随以下因素而变化：（i）所测试的损伤类型和DNA糖基化酶；（ii）局部序列背景以及相对链损伤之间的错开情况；（iii）氧化损伤相对于潜在组蛋白八聚体的螺旋方向；（iv）损伤簇与核小体边缘之间的距离。在某些情况下，BER因子与一个核小体损伤的结合似乎促进了与相对链损伤的结合。DSB的形成并不总是导致核小体解体，在某些情况下，DSB形成产生的游离DNA末端仍与组蛋白八聚体结合。这些观察结果解释了核小体的特定结构和动态特性如何有助于抑制BER产生的DSB。这些研究还表明，大多数BER产生的DSB将发生在连接DNA以及与核小体周转或重塑速率升高相关的基因组区域中。
