Biology subjects

Stivers, J. T.

Publications and source records attributed to Stivers, J. T..

5 recordsLinked to original sources

bioRxiv2026

Transition metal activation reframes SAMHD1 regulation

SAMHD1 is the lone human dNTP triphosphohydrolase and is intimately linked to HIV viral restriction, dNTP pool maintenance, resistance to chemotherapy, and the autoinflammatory Aicardi-Goutieres syndrome. While its substrate promiscuity and nucleotide basis of activity have been extensively studied, the identity and mechanistic roles of its metal cofactors remain poorly defined. Here, we integrate elemental analysis, spectroscopy, protein cross-linking, and enzyme kinetics to elucidate the molecular mechanisms underlying metal-dependent activation and catalysis in SAMHD1. Our findings establish that transition metals are essential components of SAMHD1 function, highlight their overlooked role in allosteric regulation, and reveal a central role for iron in organizing the dinuclear active site. We show that iron is preferentially incorporated in one position of the bimetallic core, where it promotes recruitment of a second divalent metal ion required for activity. While manganese can substitute for iron, it alters the metal binding equilibria, highlighting the unique functional properties of iron. Notably, SAMHD1 exhibits metal cofactor promiscuity at the second metal site, accommodating diverse divalent metals with distinct effects on activity. Cumulatively, our findings establish iron as a core structural and functional determinant of SAMHD1 catalysis and reveal how transition metal selectivity and flexibility enable enzymatic activity across diverse cellular environments and metal flux conditions. Significance StatementSAMHD1 is a central regulator of cellular dNTP poolsand an essential antiviral restriction factor, yet its metal dependence remains poorly defined. Here, we show that SAMHD1 is not a magnesium-driven enzyme but a transition-metal-dependent hydrolase in which iron plays a central structural and regulatory role. We define the metal requirements of the active and allosteric sites and demonstrate that diiron and heterodinuclear iron-containing cofactors form in solution and support catalysis. Transition metals such as iron and manganese act as more effective activators than magnesium, while plasticity at the second metal-binding site enables activity across dynamic metalation and oxidation states. SAMHD1 can thus flexibly tune antiviral defense and nucleotide metabolism, bypassing constraints imposed by metal availability and the cellular redox environment.

biochemistry↗

bioRxiv2023

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

The dNTPase activity of tetrameric SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) plays a critical role in cellular dNTP regulation. SAMHD1 also associates with stalled DNA replication forks, DNA repair foci, ssRNA, and telomeres. The above functions require nucleic acid binding by SAMHD1, which may be modulated by its oligomeric state. Here we establish that the guanine-specific A1 activator site of each SAMHD1 monomer is used to target the enzyme to guanine nucleotides within single-stranded (ss) DNA and RNA. Remarkably, nucleic acid strands containing a single guanine base induce dimeric SAMHD1, while two or more guanines with [~]20 nucleotide spacing induce a tetrameric form. A cryo-EM structure of ssRNA-bound tetrameric SAMHD1 shows how ssRNA strands bridge two SAMHD1 dimers and stabilize the structure. This ssRNA-bound tetramer is inactive with respect to dNTPase and RNase activity.

biochemistry↗

bioRxiv2022

Replication-competent HIV-1 in human alveolar macrophages and monocytes despite nucleotide pools with elevated dUTP

Although CD4+ memory T cells are considered the primary latent reservoir for HIV-1, replication competent HIV has been detected in tissue macrophages in both animal and human studies. During in vitro HIV infection, the depleted nucleotide pool and high dUTP levels in monocyte derived macrophages (MDM) leads to proviruses with high levels of dUMP, which has been implicated in viral restriction or reduced transcription depending on the uracil base excision repair (UBER) competence of the macrophage. Incorporated dUMP has also been detected in viral DNA from circulating monocytes (MC) and alveolar macrophages (AM) of HIV infected patients on antiretroviral therapy (ART), establishing the biological relevance of this phenotype but not the replicative capacity of dUMP-containing proviruses. As compared to in vitro differentiated MDM, AM from normal donors had 6-fold lower levels of dTTP and a 6-fold increased dUTP/dTTP, indicating a highly restrictive dNTP pool for reverse transcription. Expression of uracil DNA glycosylase (UNG) was 8-fold lower in AM compared to the already low levels in MDM. Accordingly, [~]80% of HIV proviruses contained dUMP, which persisted for at least 14-days due to low UNG excision activity. Unlike MDM, AM expression levels of UNG and SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) increased over 14 days post-HIV infection, while dUTP nucleotidohydrolase expression decreased. These AM-specific effects suggest a restriction response centered on excising uracil from viral DNA copies and increasing relative dUTP levels. Despite the restrictive nucleotide pools, we detected rare replication competent HIV in AM, peripheral MC, and CD4+ T cells from ART-treated donors. These findings indicate that the potential integration block of incorporated dUMP is not realized during in vivo infection of AM and MC due to the near absence of UBER activity. In addition, the increased expression of UNG and SAMHD1 in AM post-infection is too slow to prevent integration. Accordingly, dUMP persists in integrated viruses, which based on in vitro studies, can lead to transcriptional silencing. This possible silencing outcome of persistent dUMP could promote viral latency until the repressive effects of viral dUMP are reversed.

microbiology↗

bioRxiv2022

Phosphorylation of SAMHD1 Thr592 increases C-terminal domain dynamics, tetramer dissociation, and ssDNA binding kinetics

SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), enabling the enzyme to localize to stalled replication forks (RFs) and promote their restart. Since localization of a potent dNTPase at stalled RFs is not harmonious with DNA replication, we used a series of kinetic and thermodynamic measurements to explore a hypothesis where the combined effects of T592 phosphorylation and ssDNA binding serves as a dual switch to turn-off SAMHD1 dNTPase activity. We report that phosphorylation has only a small effect on the dNTPase activity and ssDNA binding affinity of SAMHD1. However, perturbation of the native T592 by phosphorylation decreased the thermal stability of tetrameric SAMHD1 and accelerated tetramer dissociation in the absence and presence of ssDNA (~15-fold). In addition, we found that ssDNA binds competitively with GTP to the A1 site. A full-length SAMHD1 cryo-EM structure revealed substantial baseline dynamics in the C-terminal domain (which contains T592) which may be modulated by phosphorylation. We propose that T592 phosphorylation increases tetramer dynamics and allows invasion of ssDNA into the A1 site and the previously characterized DNA binding surface at the dimer-dimer interface. These features are consistent with rapid and regiospecific inactivation of pSAMHD1 dNTPase at RFs or other sites of free ssDNA in cells.

biochemistry↗

bioRxiv2020

Deficient uracil base excision repair leads to persistent dUMP in HIV proviruses during infection of monocytes and macrophages

Non-dividing cells of the myeloid lineage such as monocytes and macrophages are target cells of HIV that have low dNTP pool concentrations and elevated levels of dUTP, which leads to frequent incorporation of dUMP opposite to A during reverse transcription ("uracilation"). One factor determining the fate of dUMP in proviral DNA is the host cell uracil base excision repair (UBER) system. Here we explore the relative UBER capacity of monocytes (MC) and monocyte-derived macrophages (MDM) and the fate of integrated uracilated viruses in both cell types to understand the implications of viral dUMP on HIV diversification and infectivity. We find that monocytes are almost completely devoid of functional UBER, while macrophages are mainly deficient in the initial enzyme uracil DNA glycosylase (hUNG2). Accordingly, dUMP persists in viral DNA during the lifetime of a MC and can only be removed after differentiation of MC into MDM. Overexpression of human uracil DNA glycosylase in MDM prior to infection resulted in rapid removal of dUMP from HIV cDNA and near complete depletion of dUMP-containing viral copies. This finding establishes that the low hUNG2 expression level in these cells limits UBER but that hUNG2 is restrictive against uracilated viruses. In contrast, overexpression of hUNG2 after viral integration did not accelerate the excision of uracils, suggesting that they may poorly accessible in the context of chromatin. We found that viral DNA molecules with incorporated dUMP contained unique (+) strand transversion mutations that were not observed when dUMP was absent (G[->]T, T[->]A, T[->]G, A[->]C). These observations and other considerations suggest that dUMP introduces errors predominantly during (-) strand synthesis when the template is RNA. These mutations may arise from the increased mispairing and duplex destabilizing effects of dUMP relative to dTMP during reverse transcription. Overall, the likelihood of producing a functional virus from in vitro infection of MC is about 50-fold and 300-fold reduced as compared to MDM and activated T cells. The results implicate viral dUMP incorporation in MC and MDM as a potential viral diversification and restriction pathway during human HIV infection.

microbiology↗