Molecular basis for ligation of mismatched DNA ends

描述(申请人提供)：连接错配DNA末端的分子基础。非同源末端连接(NHEJ)途径修复由电离辐射或酶切割产生的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必须在优化DNA序列保存的同时将末端连接起来。这项提案关注的是一种前所未有的连接酶活性，它可以通过错配的突出端连接DNA末端。这种不匹配的末端(Mend)连接酶活性需要Ku、XRCC4/Ligase IV和Cernunnos。通过连接所有其他连接酶不能连接的末端，修复连接酶优化了DNA序列的保存。这一非凡活动的分子基础是什么？Cernunnos和XRCC4的晶体结构和突变形式导致了一个令人惊讶的假设：Cernunnos和XRCC4形成了一条细丝，将不匹配的DNA末端排列在一起进行连接。强大的方法将被用来支持这一假说或生成另一种模型。具体目的是：目的1：研究修复连接酶复合体中的蛋白质相互作用。错掺质子-烷基交换(MPAX)将应用于Mend连接酶蛋白。MPAX随机将半胱氨酸错误结合到目标蛋白中，然后通过烷基化识别暴露的半胱氨酸残基。这将允许高通量绘制被蛋白质-蛋白质相互作用掩埋的暴露残基的地图。目的2：确定突变对NHEJ的影响。将合成突变蛋白，并检测Mend连接酶活性、提取物中的NHEJ和细胞中的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修复连接酶中产生明显的生化异常。目的3：分析修复连接酶复合体在DNA末端的组装情况。为了通过修复连接酶来检测DNA末端的排列，我们将测试各种错配的突出物。EMSA将决定Ku是否稳定Cernunnos和XL与DNA的结合。光交联将决定蛋白质在DNA末端的位置。下拉分析将测试修复连接酶是否促进末端的突触。初步的电子显微镜图像显示自发形成了XRCC4/Cernunnos丝状结构，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有影响。Ligase IV和Cernunnos基因的突变会导致免疫缺陷和癌症易感性的遗传性疾病。环境中的DSB会致癌。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。与公共卫生相关：这项提议旨在了解非同源末端连接，这是一种将断裂的染色体重新连接的途径。这种中断发生在辐射后或在产生对感染的免疫力的过程中。这一途径的核心反应可以连接受损的DNA末端，即使传统的酶不能。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.