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 G0G1y 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 G0G1y transition in MDM. (ii) Measure the repair capacities of homogenous G0 and G1y MDM populations and the fate of genomic uracils after the G0G1y 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.

Public Health Relevance

This proposal will elucidate how long-lived, non-dividing macrophage immune cells repair genomic DNA by reversibly transitioning between a repair deficient resting state and a repair competent active state. The coordinated transition between these states is implicated in macrophage dysfunction and susceptibility to viral infection. !

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function A Study Section (MSFA)
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Marino, Pamela
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Johns Hopkins University
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Weiser, Brian P; Rodriguez, Gaddiel; Cole, Philip A et al. (2018) N-terminal domain of human uracil DNA glycosylase (hUNG2) promotes targeting to uracil sites adjacent to ssDNA-dsDNA junctions. Nucleic Acids Res 46:7169-7178
Rodriguez, Gaddiel; Esadze, Alexandre; Weiser, Brian P et al. (2017) Disordered N-Terminal Domain of Human Uracil DNA Glycosylase (hUNG2) Enhances DNA Translocation. ACS Chem Biol 12:2260-2263
Weiser, Brian P; Stivers, James T; Cole, Philip A (2017) Investigation of N-Terminal Phospho-Regulation of Uracil DNA Glycosylase Using Protein Semisynthesis. Biophys J 113:393-401
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Cravens, Shannen L; Stivers, James T (2016) Comparative Effects of Ions, Molecular Crowding, and Bulk DNA on the Damage Search Mechanisms of hOGG1 and hUNG. Biochemistry 55:5230-42
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Cravens, Shannen L; Schonhoft, Joseph D; Rowland, Meng M et al. (2015) Molecular crowding enhances facilitated diffusion of two human DNA glycosylases. Nucleic Acids Res 43:4087-97
Seamon, Kyle J; Sun, Zhiqiang; Shlyakhtenko, Luda S et al. (2015) SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity. Nucleic Acids Res 43:6486-99

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