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）途径修复由电离辐射或酶促裂解产生的DNA双链断裂（DSB）以产生免疫多样性。为了确保染色体的完整性，NHEJ必须连接末端，同时优化DNA序列的保存。这项提议集中在一种前所未有的连接酶活性上，这种活性可以将DNA末端与错配的突出端连接起来。这种错配末端（MEnd）连接酶活性需要Ku、XRCC 4/连接酶IV和Cernunnos。通过连接对所有其他连接酶都不敏感的末端，MEnd连接酶优化了DNA序列的保存。这种非凡的活性的分子基础是什么？Cernunnos和XRCC 4的晶体结构和突变形式导致了一个令人惊讶的假设：Cernunnos和XRCC 4形成了一个细丝，可以对齐不匹配的DNA末端进行连接。将使用强大的方法来支持假设或生成替代模型。具体的目的是：目的1：表征蛋白质的相互作用在MEND连接酶复合物。错误掺入质子-烷基交换（MPAX）将应用于MEd连接酶蛋白。MPAX随机地将半胱氨酸错误地掺入到靶蛋白中，然后通过烷基化来识别暴露的半胱氨酸残基。这将允许通过蛋白质-蛋白质相互作用掩埋的暴露残基的高通量映射。目的2：确定突变对NHEJ的影响。将合成突变蛋白，并检测MEd连接酶活性、提取物中的NHEJ和细胞中的V（D）J重组。Cernunnos和连接酶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，而另一些则会导致严重的联合免疫缺陷。我们将确定这种突变是否会在MEND连接酶中产生明显的生化异常。目的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，我们将测试各种错配的突出端。EMSA将确定Ku是否稳定Cernunnos和XL与DNA的结合。光交联将确定蛋白质在DNA末端的位置。下拉测定将测试MEd连接酶是否促进末端的突触。初步的电子显微镜图像表明自发形成XRCC 4/Cernunnos丝状结构，我们将确定这些结构是否是假设的丝状体。我们的研究对人类健康有影响。连接酶IV和Cernunnos基因的突变产生免疫缺陷和癌症易感性的遗传性疾病。环境DSB可导致癌症。许多有效的抗癌剂产生DSB。因此，了解修复DSB的分子基础可能会改善癌症预防和治疗。公共卫生相关性：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。这种断裂发生在辐射后或产生抗感染免疫力的过程中。该途径的核心反应可以连接受损的DNA末端，即使常规酶不能。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.