Molecular basis for ligation of mismatched DNA ends

描述（由申请人提供）：错配 DNA 末端连接的分子基础。非同源末端连接 (NHEJ) 途径修复电离辐射或酶裂解产生的 DNA 双链断裂 (DSB)，以产生免疫多样性。为了确保染色体完整性，NHEJ 必须连接末端，同时优化 DNA 序列的保存。该提案重点关注前所未有的连接酶活性，该活性可以连接具有不匹配突出端的 DNA 末端。这种错配末端 (MEnd) 连接酶活性需要 Ku、XRCC4/Ligase IV 和 Cernunnos。 MEnd 连接酶通过连接所有其他连接酶均难处理的末端，优化 DNA 序列的保存。这种非凡活性的分子基础是什么？ Cernunnos 和 XRCC4 的晶体结构和突变形式引发了一个令人惊讶的假设：Cernunnos 和 XRCC4 形成一根细丝，将不匹配的 DNA 末端对齐以进行连接。将使用强大的方法来支持假设或生成替代模型。具体目标是： 目标 1：表征 MEnd 连接酶复合物中的蛋白质相互作用。错误掺入质子烷基交换 (MPAX) 将应用于 MEnd 连接酶蛋白。 MPAX 将半胱氨酸随机错误地掺入目标蛋白中，然后通过烷基化识别暴露的半胱氨酸残基。这将允许对因蛋白质-蛋白质相互作用而隐藏的暴露残基进行高通量绘图。目标 2：确定突变对 NHEJ 的影响。将合成突变蛋白并测试 MEnd 连接酶活性、提取物中的 NHEJ 以及细胞中的 V(D)J 重组。 Cernunnos 和 Ligase IV 的临床突变赋予细胞放射敏感性。然而，一些突变会导致小头畸形和生长迟缓，而另一些突变则会导致严重的联合免疫缺陷。我们将确定此类突变是否会在 MEnd 连接酶中产生明显的生化异常。目标 3：分析 MEnd 连接酶复合物在 DNA 末端的组装。为了通过 MEnd 连接酶探测 DNA 末端的比对，我们将测试各种不匹配的突出端。 EMSA 将确定 Ku 是否稳定 Cernunnos 和 XL 与 DNA 的结合。光交联将确定 DNA 末端蛋白质的位置。 Pull-down 测定将测试 MEnd 连接酶是否促进末端突触。初步的电子显微镜图像表明 XRCC4/Cernunnos 丝状结构的自发形成，我们将确定这些结构是否是假设的丝状结构。我们的研究对人类健康具有影响。连接酶 IV 和 Cernunnos 基因的突变会导致免疫缺陷和癌症易感性等遗传性疾病。环境 DSB 可能导致癌症。许多有效的抗癌药物都会产生 DSB。因此，了解修复 DSB 的分子基础可能会改善癌症的预防和治疗。公共健康相关性：该提案旨在了解非同源末端连接，这是一种重新连接断裂染色体的途径。这种中断发生在辐射后或产生针对感染的免疫力的过程中。该途径的核心反应可以连接受损的 DNA 末端，即使传统酶无法连接。核心反应的缺陷会导致免疫缺陷、生长迟缓和癌症。

DESCRIPTION (provided by applicant): Molecular basis for ligation of mismatched DNA ends. The nonhomologous end joining (NHEJ) pathway repairs DNA double-strand breaks (DSBs) produced by ionizing radiation or by enzymatic cleavage to generate immunological diversity. To ensure chromosomal integrity, NHEJ must join the ends while optimizing the preservation of DNA sequence. This proposal focuses on an unprecedented ligase activity that can join DNA ends with mismatched overhangs. This mismatched end (MEnd) ligase activity requires Ku, XRCC4/Ligase IV and Cernunnos. By ligating ends refractory to all other ligases, MEnd ligase optimizes preservation of DNA sequence. What is the molecular basis for this remarkable activity? Crystal structures and mutant forms of Cernunnos and XRCC4 led to a surprising hypothesis: Cernunnos and XRCC4 form a filament that aligns mismatched DNA ends for ligation. Powerful methods will be used to either support the hypothesis or generate an alternative model. The specific aims are to: Aim 1: Characterize protein interactions in the MEnd ligase complex. Misincorporation proton-alkyl exchange (MPAX) will be applied to the MEnd ligase proteins. MPAX randomly misincorporates cysteines into the targeted protein, and then identifies exposed cysteine residues by alkylation. This will permit high-throughput mapping of exposed residues that are buried by protein-protein interactions. Aim 2: Determine the effect of mutations on NHEJ. Mutant proteins will be synthesized and tested for MEnd ligase activity, NHEJ in extracts and V(D)J recombination in cells. Clinical mutations in Cernunnos and Ligase IV confer cellular radiosensitivity. However, some of the mutations cause microcephaly and growth delay, while others cause severe combined immunodeficiency. We will determine if such mutations produce distinct biochemical abnormalities in MEnd ligase. Aim 3: Analyze the assembly of the MEnd ligase complex on DNA ends. To probe alignment of DNA ends by MEnd ligase, we will test various mismatched overhangs. EMSAs will determine whether Ku stabilizes binding of Cernunnos and XL to DNA. Photo-cross-linking will determine the positions of proteins at the DNA ends. Pull-down assays will test whether MEnd ligase facilitates synapsis of ends. Preliminary electron microscopy images suggest spontaneous formation of XRCC4/Cernunnos filament- like structures, and we will determine whether these structures are the hypothesized filament. Our studies have implications for human health. Mutations in the Ligase IV and Cernunnos genes produce inherited diseases of immune deficiency and cancer susceptibility. Environmental DSBs can cause cancer. Many effective anticancer agents generate DSBs. Therefore, understanding the molecular basis for repairing DSBs may lead to improvements in cancer prevention and treatment. PUBLIC HEALTH RELEVANCE: This proposal aims to understand nonhomologous end-joining, a pathway that rejoins broken chromosomes. Such breaks occur after radiation or during the process that produces immunity against infection. The core reaction for this pathway can join damaged DNA ends, even when conventional enzymes cannot. Defects in the core reaction lead to immune deficiency, growth retardation, and cancer.