SAMHD1 promotes DNA end resection to facilitate DNA repair by homologous recombination

DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV- 1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity

SAMHD1-mediated dNTP pool regulation in macrophage : effect on HIV-1 reverse transcription kinetics and fidelity

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2014.HIV-1 replication is restricted in macrophages as compared to activated CD4+ T cells. Recently, SAMHD1 has been identified as a HIV-1 restriction factor that is counteracted by Vpx. Here we show that HIV-1 infection of monocyte-derived macrophages (MDM) is restricted due to active hydrolyzation of dNTP by SAMHD1, reducing the cellular dNTP pool below the Km and Kd of HIV-1 reverse transcriptase (RT), possibly slowing proviral DNA synthesis. MDMs treatment by exogenous dNs or VLPs containing Vpx elevated cellular dNTP pools, suggesting that both affect similar pathway. Together, these observations showed that SAMHD1 is actively involved in regulation of dNTPs and contributes to the low dNTP concentration found in macrophages. RT can synthesize proviral DNA in non-dividing cells like macropages due to its tight dNTP binding affinity. A structural model shows that the A114 occupies a dNTP binding pocket. Thus, we tested whether increasing size of the side chain at position 114 will reduce RT dNTP binding affinity by reducing space in the pocket. Indeed, our biochemical data demonstrate that the residue 114 mutant RTs have reduced polymerase activity at low dNTP concentrations. Pre-steady state kinetic study also shows that the A114C RT mutant has reduced dNTP binding affinity (Kd), but without altering catalysis (kpol). Compared to WT, the A114C mutant RT showed similar incorporation capability of acydNTPs lacking the sugar ring. These data support that A114 plays a key role in the tight dNTP binding affinity of RT and a unique HIV-1 infectivity of macrophages. RT incorporates rNTPs frequently under macrophage dNTP conditions. Thus we investigated the mechanistic impact of rNMP embedded in DNA templates on RT-mediated DNA synthesis. Our study showed that under simulated macrophage dNTP concentrations, rNMP induced pausing of RT and delayed DNA synthesis kinetics. Although RT template/primer (T/P) binding affinity was not altered, its dNTP incorporation kinetics was significantly reduced at one nucleotide upstream and downstream of the rNMP site. RT becomes more error prone with an elevated mismatch extension capability but not enhanced misinsertion capability. These data show that rNMPs embedded in DNA templates may influence HIV-1 reverse transcription kinetics and simultaneously viral mutagenesis

Viral Particle-Mediated SAMHD1 Depletion Sensitizes Refractory Glioblastoma to DNA-Damaging Therapeutics by Impairing Homologous Recombination

The current standard-of-care treatment for glioblastoma includes DNA damaging agents, γ-irradiation (IR) and temozolomide (TMZ). These treatments fail frequently and there is limited alternative strategy. Therefore, identifying a new therapeutic target is urgently needed to develop a strategy that improves the efficacy of the existing treatments. Here, we report that tumor samples from GBM patients express a high level of SAMHD1, emphasizing SAMHD1’s importance. The depletion of SAMHD1 using virus-like particles containing Vpx, VLP(+Vpx), sensitized two independent GBM cell lines (LN-229 and U-87) to veliparib, a well-established PARP inhibitor, and slowed cell growth in a dose-dependent manner. In the mouse GBM xenograft model, Vpx-mediated SAMHD1 depletion reduced tumor growth and SAMHD1 knockout (KO) improved survival. In combination with IR or TMZ, SAMHD1 KO and exposure to 50% growth inhibitory dose (gID50) of VLP(+Vpx) displayed a synergistic effect, resulting in impaired HR, and improved LN-229 cells’ sensitivity to TMZ and IR. In conclusion, our finding demonstrates that SAMHD1 promotes GBM resistance to treatment, and it is a plausible therapeutic target to improve the efficacy of TMZ and IR in GBM. Furthermore, we show that Vpx could be a potential therapeutic tool that can be utilized to deplete SAMHD1 in GBM

Determining cell viability.

<p>A) 1×10⁶ monocytes/well were plated and then left untreated (control), GM-CSF-treated or <i>Leishmania</i>-infected at an MOI = 7. At various days afterwards, three wells/condition were collected, pooled and the cell numbers determined. Nine independent donors were examined. B–D) Images were captured using bright field microscope. Control cells had very few adherent cells as compared to GM-CSF-treated and <i>Leishmania</i>-infected cell cultures.</p

Leishmania Induces Survival, Proliferation and Elevated Cellular dNTP Levels in Human Monocytes Promoting Acceleration of HIV Co-Infection

<div><p>Leishmaniasis is a parasitic disease that is widely prevalent in many tropical and sub-tropical regions of the world. Infection with <em>Leishmania</em> has been recognized to induce a striking acceleration of Human Immunodeficiency Virus Type 1 (HIV-1) infection in coinfected individuals through as yet incompletely understood mechanisms. Cells of the monocyte/macrophage lineage are the predominant cell types coinfected by both pathogens. Monocytes and macrophages contain extremely low levels of deoxynucleoside triphosphates (dNTPs) due to their lack of cell cycling and S phase, where dNTP biosynthesis is specifically activated. Lentiviruses, such as HIV-1, are unique among retroviruses in their ability to replicate in these non-dividing cells due, at least in part, to their highly efficient reverse transcriptase (RT). Nonetheless, viral replication progresses more efficiently in the setting of higher intracellular dNTP concentrations related to enhanced enzyme kinetics of the viral RT. In the present study, <em>in vitro</em> infection of CD14+ peripheral blood-derived human monocytes with <em>Leishmania major</em> was found to induce differentiation, marked elevation of cellular p53R2 ribonucleotide reductase subunit and R2 subunit expression. The R2 subunit is restricted to the S phase of the cell cycle. Our dNTP assay demonstrated significant elevation of intracellular monocyte-derived macrophages (MDMs) dNTP concentrations in <em>Leishmania</em>-infected cell populations as compared to control cells. Infection of <em>Leishmania</em>-maturated MDMs with a pseudotyped GFP expressing HIV-1 resulted in increased numbers of GFP+ cells in the <em>Leishmania</em>-maturated MDMs as compared to control cells. Interestingly, a sub-population of <em>Leishmania</em>-maturated MDMs was found to have re-entered the cell cycle, as demonstrated by BrdU labeling. In conclusion, <em>Leishmania</em> infection of primary human monocytes promotes the induction of an S phase environment and elevated dNTP levels with notable elevation of HIV-1 expression in the setting of coinfection.</p> </div

HIV-1 RT based dNTP assay.

<p>A) Diagram shows how a single nucleotide extension assay is done. The reaction contains the template, 5′ ³²P-end-labeled primer, HIV-1 RT and cellular dNTP extract. After the reactions are completed, they are resolved on a polyacrylamide gel to determine product formation. The concentrations of dGTP and dTTP in the cellular extract will determine the amount of primer extension. B) For the different groups, primer extension products are shown for day 7 and day 13 cellular extracts. Control monocytes, GM-CSF-treated and <i>Leishmania</i>-infected cells are shown. The GM-CSF-treated cells are used as a positive control. Primer only (unextended) is indicated with an open arrow, whereas extended product formation is indicated with a filled arrow. The negative controls contain no dNTPs and are shown in lane 1. Positive controls (lanes 2) contained 50 µM of exogenous dNTP mix. Lanes 3–8 are cellular extracts from the different treatment groups. C) Graphs plotting the percent extension of dGTP and dTTP. From the primer extension assays, data was plotted for days 7 and 13 for dGTP (n = 9; top graphs) and for dTTP (n = 6; bottom graphs). Significantly different groups are displayed as * = p<0.05, ** = p<0.01 and *** = p<0.001 for the different groups as compared to control monocytes.</p

HIV-1 D3 vector analysis.

<p>A) Control, GM-CSF-treated and <i>Leishmania</i>-infected cells were transduced with HIV-1 D3 GFP vector. Twenty-four hours after transduction, the cells were examined for GFP expression (top row). Very few monocytes were GFP+, whereas more cells were GFP+ for the GM-CSF-treated and <i>Leishmania</i>-infected groups. For this experiment, the <i>L. major</i> were labeled with PKH dye and showed that the monocytes were infected. Bright field images were captured for the different groups. B) FACS analysis was done of three different cell populations. Data is representative of four different donors done at 24 h. Complete data sets for 24 h and 48 h are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002635#ppat-1002635-t001" target="_blank">Table 1</a>.</p

Percent HIV-1 D3 GFP Transduction.

<p>Percent HIV-1 D3 GFP Transduction.</p

Western blot analysis of ribonucleotide reductase.

<p>A) Cellular lysates from different treatment groups were analyzed for ribonucleotide reductase R2 and p53R2 subunits. Afterwards, blots were stripped and re-probed for actin. Freshly isolated monocytes (Mo), day 13 maturated GM-CSF (GM-CSF) and <i>Leishmania</i> (Leish) MDMs are shown. B) Quantitiation of western blots was done. Freshly isolated monocytes were set to 1 and increases in R2 and p53R2 expression levels for GM-CSF- and <i>Leishmania</i>-maturated MDMs groups are shown. Mean and SEM are displayed for four independent donors. C) qRT-PCR analysis was done on total cellular RNA extracts. mRNA fold changes for the different treatment groups (n = 3) are graphed as mean and SEM. Significantly different groups (p<0.05) as compared to monocyte control group are indicated with an asterisk (*).</p

2LTR circle copy number ratio.

<p>The different treatment groups were treated with the D3 GFP vector. At 48 h after transduction, total cellular DNA was collected and analyzed by real-time PCR. The control group for each donor was set to 1 and then compared to GM-CSF- and <i>Leishmania</i>-maturated MDMs groups. Mean and SEM are plotted. The significantly different group (p<0.05), as compared to control, is indicated with an asterisk. Seven independent donors were analyzed.</p