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

通过可逆的伪 G1 细胞周期转变对非分裂巨噬细胞进行 DNA 修复

• 批准号: 10247072
• 负责人: JAMES T. STIVERS
• 金额: $ 45.85万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10247072
• 关键词: Agonist; Autologous; Base Excision Repairs; Base Pairing; Biochemical; Biological Assay; Blood Coagulation Factor VII; Cell Cycle; Cells; Chromatin; Complementary DNA; Conflict (Psychology); DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA biosynthesis; Data; Deamination; Environment; Excision; Fractionation; Frequencies; Functional disorder; Genome; Genomic DNA; Genomics; Glean; HIV; HIV Infections; Histone Deacetylase Inhibitor; Human; Immune; Infection; Inflammatory Response; Interphase Cell; Investigation; Knowledge; MEKs; Maps; Measures; Metabolism; Methods; Minor; Mutation; Nucleotides; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphotransferases; Poly(ADP-ribose) Polymerases; Polymerase; Population; Predisposition; Process; Proviruses; Reporter; Reporter Genes; Resolution; Rest; Reverse Transcription; Role; Serum; Single Strand Break Repair; Site; Thymidine; Time; Uracil; Viral; Viral Proteins; Virus; Virus Diseases; base; fetal bovine serum; inhibitor/antagonist; macrophage; oxidation; receptor; repaired; sequencing platform; small molecule; stem; tool; uracil-DNA glycosylase; vascular inflammation

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.

所有忠实的DNA修复过程都需要平衡的dNTP池。符合此要求的，非分割的 巨噬细胞已经耗尽了典型的dNTPs和高水平的诱变性dUTP，dUTP被用作防御 这不仅是对抗病毒感染的策略，而且对宿主基因组的完整性构成威胁。这项建议会 探索基因组保真度如何在具有耗尽和促突变dNTP的巨噬细胞中维持 池此外，从这项巨噬细胞DNA修复研究中获得的知识将用于专门研究 靶向HIV前病毒，其含有高水平的dUMP，所述dUMP由逆转录过程中dUTP的存在产生 在巨噬细胞中转录。这一建议的基础源于我们的发现，非分裂巨噬细胞 (MDM)存在两个不同的群体，就其dUTP池而言，尿嘧啶碱基切除修复（UBER） 艾滋病毒感染状况和易感性。这两个种群是偶然发现的， 在用HIV假病毒构建体感染期间分离为GFP-（高dUTP）和GFP+（低dUTP 含有GFP报告基因。进一步的研究将GFP-群体表征为静息（G0）， GFP+群体为假G1（G1 y），能够进行低水平的DNA复制，但不能进行细胞分裂。因此，在本发明中， 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， 进入G1y状态，具有有利于高保真DNA修复的环境。在三个具体目标中，我们 建议：（i）确定刺激MDM中G0 <$G1 y转换的血清因子。利用逆转录病毒 GFP报告基因检测，我们将使用经典的生化分离方法来分离血清小分子 刺激MDM中的G0àG1y转变。(ii)测量同质G0和G1y的修复能力 MDM人群和基因组尿嘧啶在G0àG1y转换后的命运。我们将在地图上的尿嘧啶 通过开发第一个能够区分尿嘧啶和 单核苷酸解析（U2C-Seq）。这些基因组尿嘧啶的命运（修复， 突变，链断裂）将在转变为G1y状态后确定。(iii)药理学上破坏 尿嘧啶化的HIV前病毒在G0 MDM中减少病毒相关的炎症反应。新数据显示 HDAC抑制剂可以诱导染色质开放，并将隔离的尿嘧啶化前病毒暴露于尿嘧啶 切除在一项新的艾滋病毒靶向策略中，我们建议使用小分子药物阻断UBER的切除后阶段。 分子。该策略利用HDAC抑制剂使难以接近的尿嘧啶暴露于切除 通过尿嘧啶DNA糖基化酶，和第二种药物，抑制所产生的毒性脱碱基位点的修复。的 功能性病毒的减少将与病毒蛋白质表达的减少和病毒蛋白质表达的减少相关。 MDM中的炎症反应。因此，这项建议将阐明巨噬细胞免疫细胞如何修复 通过在修复缺陷的静息状态和修复活性之间可逆地转变， 状态以及这种转变在巨噬细胞功能障碍和对病毒感染的易感性中的作用。

项目成果

Single-Stranded Nucleic Acids Bind to the Tetramer Interface of SAMHD1 and Prevent Formation of the Catalytic Homotetramer.

单链核酸与 SAMHD1 的四聚体界面结合并阻止催化同源四聚体的形成。

• DOI: 10.1021/acs.biochem.6b00986
• 发表时间: 2016-11-08
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Seamon KJ;Bumpus NN;Stivers JT
• 通讯作者: Stivers JT

Guanine-containing ssDNA and RNA induce dimeric and tetrameric structural forms of SAMHD1.

• DOI: 10.1093/nar/gkad971
• 发表时间: 2023-12-11
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Orris, Benjamin;Sung, Min Woo;Bhat, Shridhar;Xu, Yingrong;Huynh, Kevin W.;Han, Seungil;Johnson, Darren C.;Bosbach, Benedikt;Shields, David J.;Stivers, James T.
• 通讯作者: Stivers, James T.

Guanine-containing ssDNA and RNA induce dimeric and tetrameric SAMHD1 in cryo-EM and binding studies.

在冷冻电镜和结合研究中，含鸟嘌呤的 ssDNA 和 RNA 诱导二聚体和四聚体 SAMHD1。

• DOI: 10.1101/2023.06.15.544806
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Orris,Benjamin;Sung,MinWoo;Bhat,Shridhar;Xu,Yingrong;Huynh,KevinW;Han,Seungil;Johnson,DarrenC;Bosbach,Benedikt;Shields,DavidJ;Stivers,JamesT
• 通讯作者: Stivers,JamesT

SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity.

• DOI: 10.1093/nar/gkv633
• 发表时间: 2015-07-27
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Seamon KJ;Sun Z;Shlyakhtenko LS;Lyubchenko YL;Stivers JT
• 通讯作者: Stivers JT

Small molecule versus DNA repair nanomachine.

小分子与 DNA 修复纳米机器。

• DOI: 10.1038/nchembio0208-86
• 发表时间: 2008
• 期刊: Nature chemical biology
• 影响因子: 14.8
• 作者: Stivers,JamesT