DNA Repair in Non-Dividing Macrophages Through Reversible Go to pseudo-G1 Cell Cycle Transitions

所有忠实的DNA修复过程都需要平衡的dNTP池。与此要求相冲突，不分彼此 巨噬细胞耗尽了典型的dNTP和高水平的诱变dUTP，这是一种用于防御的 对抗病毒感染的策略，但也对宿主基因组的完整性构成威胁。这项提议将 探索如何在具有耗尽和促突变dNTP的巨噬细胞中保持基因组的保真度 游泳池。此外，从巨噬细胞DNA修复研究中获得的知识将被用于特定的 在反向过程中由于存在dUTP而导致包含高水平转储的目标HIV前病毒 巨噬细胞的转录。这一建议的基础源于我们的发现，未分裂的巨噬细胞 (MDM)作为两个不同的群体存在，相对于其dUTP池，尿嘧啶碱基切除修复(Uber) 艾滋病毒感染的状况和易感性。这两个种群被偶然发现是因为他们 在感染HIV伪病毒时分离为GFP-(高dUTP)和GFP+(低dUTP) 含有绿色荧光蛋白报告基因。进一步的研究表明，绿色荧光蛋白的种群处于休眠状态(G0)和 GFP+群体为伪G1(G1y)，能够低水平复制DNA，但不能进行细胞分裂。因此， 一种解决修复悖论的机制正在出现，其中巨噬细胞可以被刺激到可逆地 进入一个具有有利于高保真DNA修复的环境的G1y状态。在三个具体目标中，我们 建议：(I)确定刺激MDM G0-G1y转变的血清因素。利用逆转录病毒 GFP报告分析，我们将使用经典的生化分离方法来分离血清小分子 这刺激了MDM的G0àG1y转变。(Ii)测定同质G0和G1y的修复能力 MDM种群和基因组尿囊菌在G0àG1y转变后的命运。我们将在 建立首个区分尿嘧啶和尿嘧啶的测序平台 单核苷酸拆分DNA中的胸苷(U2C-Seq)。这些基因组尿嘧啶的命运(修复， 突变、链断裂)将在转换到G1y状态后确定。(三)药物性销毁 尿化艾滋病毒在G0 MDM中传播，以减少病毒相关的炎症反应。新数据表明 HDAC抑制剂可诱导染色质开放并使隔离的尿嘧啶前病毒暴露于尿嘧啶 切除。在一项针对艾滋病毒的新战略中，我们提议使用小剂量的药物来阻止优步的切除后阶段 分子。这一策略利用了一种HDAC抑制剂，使无法接触的尿嘧啶暴露在切除后 通过尿嘧啶DNA糖基酶，以及第二种药物，抑制由此产生的有毒碱性部位的修复。这个 功能性病毒的减少将与病毒蛋白表达的减少和 MDM的炎症反应。这一建议将阐明巨噬细胞免疫细胞如何修复 通过在修复缺陷静息状态和修复能力活性之间的可逆转换来实现基因组DNA 这种转变在巨噬细胞功能障碍和对病毒感染的易感性方面的状态和作用。

All faithful DNA repair processes require balanced dNTP pools. Conflicting with this requirement, non-dividing macrophages have depleted canonical dNTPs and high levels of mutagenic dUTP, which is used as a defense strategy against viral infection, but also poses a threat to the integrity of the host genome. This proposal will explore how genome fidelity is maintained in macrophages that have a depleted and pro-mutagenic dNTP pool. In addition, the knowledge gleaned from this study of macrophage DNA repair will be used to specifically target HIV proviruses that contain high levels of dUMP arising from the presence of dUTP during reverse transcription in macrophages. The basis of this proposal stems from our finding that non-dividing macrophages (MDM) exist as two distinct populations with respect to their dUTP pools, uracil base excision repair (UBER) status and susceptibility to HIV infection. The two populations were serendipitously detected because they segregated as GFP- (high dUTP) and GFP+ (low dUTP) during infection with an HIV pseudo-viral construct containing a GFP reporter gene. Further investigation characterized the GFP- population as resting (G0) and the GFP+ population as pseudo-G1 (G1y), capable of low levels of DNA replication, but not cell division. Thus, a mechanism to resolve the repair paradox is emerging where macrophages can be stimulated to reversibly enter a G1y-state that has an environment conducive to high-fidelity DNA repair. In three specific aims, we propose to: (i) Identify the serum factor that stimulates the G0àG1y transition in MDM. Using the retroviral GFP reporter assay, we will use classic biochemical fractionation methods to isolate the serum small molecule that stimulates the G0àG1y transition in MDM. (ii) Measure the repair capacities of homogenous G0 and G1y MDM populations and the fate of genomic uracils after the G0àG1y transition. We will map uracilation in the genomic DNA of G0 MDM by developing the first sequencing platform capable of distinguishing uracil from thymidine in DNA with single nucleotide resolution (U2C-Seq). The fate of these genomic uracils (repair, mutation, strand breaks) will be determined after transitioning to the G1y state. (iii) Pharmacologically destroy uracilated HIV proviruses in G0 MDM to reduce virus-associated inflammatory responses. New data indicates that HDAC inhibitors can induce chromatin opening and expose sequestered uracilated proviruses to uracil excision. In a new HIV targeting strategy, we propose to block the post-excision stages of UBER using small molecules. This strategy takes advantage of an HDAC inhibitor that exposes inaccessible uracils to excision by uracil DNA glycosylase, and a second drug that inhibits repair of the resultant toxic abasic sites. The decrease in functional virus will be correlated with reduced expression of viral proteins and a reduced inflammatory response in MDM. This proposal will thus elucidate how macrophage immune cells repair genomic DNA by reversibly transitioning between a repair deficient resting state and a repair competent active state and the role of this transition in macrophage dysfunction and susceptibility to viral infection.