A CRISPR/Cas9 approach reveals that the polymerase activity of DNA polymerase ß is dispensable for HIV-1 infection in dividing and nondividing cells.

Retrovirus integration into the host genome relies on several host enzymes, potentially including DNA polymerase ß (Pol ß). However, whether human Pol ß is essential for lentivirus replication in human cells is unclear. Here, we abolished DNA polymerase ß (Pol ß) expression by targeting its DNA polymerase domain with CRISPR/Cas9 in human monocytic THP-1 cells to investigate the role of Pol ß in HIV-1 transduction in both dividing and nondividing macrophage stages of THP-1 cells. Pol ß-knock-out was confirmed by enhanced sensitivity to methyl methanesulfonate-induced DNA damage. Of note, nuclear extracts from Pol ß-knock-out THP-1 cells prepared from both dividing and nondividing stages displayed significantly reduced capability to repair the gapped HIV-1 integration intermediate DNA substrate in a biochemical simulation. However, nuclear extract from both dividing and nondividing stages of the Pol ß-KO cells had detectable gap repair activity, suggesting that other host DNA polymerases also repair gapped HIV-1 DNA, particularly in dividing cells. Next, when we compared transduction using HIV-1 and simian immunodeficiency virus in control and Pol ß-KO cells, the loss of the Pol ß expression did not affect transduction efficiency of these lentiviruses in both dividing and nondividing stages. Finally, the gap repair assay indicated that limited cellular dNTP pools, but not Pol ß expression, are a primary factor for HIV-1 DNA gap repair, particularly in nondividing cells. These data support the idea that Pol ß polymerase activity is dispensable for HIV-1 infection in both dividing and nondividing stages of human cells targeted by the virus.

AKT1, LKB1, and YAP1 Revealed as MYC Interactors with NanoLuc-Based Protein-Fragment Complementation Assay.

The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. In this study, we report the development of a NanoLuc-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells. The NanoPCA system was configured to enable detection of protein-protein interactions (PPI) at the endogenous level, as shown with PRAS40 dimerization, and detection of weak interactions, such as PINCH1-NCK2. Importantly, NanoPCA allows the study of PPI dynamics with reversible interactions. To demonstrate its utility for large-scale PPI detection in mammalian intracellular environment, we have used NanoPCA to examine MYC interaction with 83 cancer-associated proteins in live cancer cell lines. Our new MYC PPI data confirmed known MYC-interacting proteins, such as MAX, GSK3A, and SMARCA4, and revealed a panel of novel MYC interaction partners, such as RAC-α serine/threonine-protein kinase (AKT)1, liver kinase B (LKB)1, and Yes-associated protein (YAP)1. The MYC interactions with AKT1, LKB1, and YAP1 were confirmed by coimmunoprecipitation of endogenous proteins. Importantly, AKT1, LKB1, and YAP1 were able to activate MYC in a transcriptional reporter assay. Thus, these vital growth control proteins may represent promising MYC regulators, suggesting new mechanisms that couple energetic and metabolic pathways and developmental signaling to MYC-regulated cellular programs.

Evidence of Kinetic Cooperativity in Dimeric Ketopantoate Reductase from Staphylococcus aureus.

Ketopantoate reductase (KPR) catalyzes the NADPH-dependent production of pantoate, an essential precursor in the biosynthesis of coenzyme A. Previous structural studies have been limited to Escherichia coli KPR, a monomeric enzyme that follows a sequential ordered mechanism. Here we report the crystal structure of the Staphylococcus aureus enzyme at 1.8 Å resolution, the first description of a dimeric KPR. Using sedimentation velocity analysis, we show that the S. aureus KPR dimer is stable in solution. In fact, our structural analysis shows that the dimeric assembly we identify is present in the majority of KPR crystal structures. Steady state analysis of S. aureus KPR reveals strong positive cooperativity with respect to NADPH (Hill coefficient of 2.5). In contrast, high concentrations of the substrate ketopantoate (KP) inhibit the activity of the enzyme. These observations are consistent with a random addition mechanism in which the initial binding of NADPH is the kinetically preferred path. In fact, Förster resonance energy transfer studies of the equilibrium binding of NADPH show only a small degree of cooperativity between subunits (Hill coefficient of 1.3). Thus, the apparently strong cooperativity observed in substrate saturation curves is due to a kinetic process that favors NADPH binding first. This interpretation is consistent with our analysis of the A181L substitution, which increases the Km of ketopantoate 844-fold, without affecting kcat. The crystal structure of KPRA181L shows that the substitution displaces Ser239, which is known to be important for the binding affinity of KP. The decrease in KP affinity would enhance the already kinetically preferred NADPH binding path, making the random mechanism appear to be sequentially ordered and reducing the kinetic cooperativity. Consistent with this interpretation, the NADPH saturation curve for KPRA181L is hyperbolic.

Substrates and Inhibitors of SAMHD1.

SAMHD1 hydrolyzes 2'-deoxynucleoside-5'-triphosphates (dNTPs) into 2'-deoxynucleosides and inorganic triphosphate products. In this paper, we evaluated the impact of 2' sugar moiety substitution for different nucleotides on being substrates for SAMHD1 and mechanisms of actions for the results. We found that dNTPs ((2'R)-2'-H) are only permissive in the catalytic site of SAMHD1 due to L150 exclusion of (2'R)-2'-F and (2'R)-2'-OH nucleotides. However, arabinose ((2'S)-2'-OH) nucleoside-5'-triphosphates analogs are permissive to bind in the catalytic site and be hydrolyzed by SAMHD1. Moreover, when the (2'S)-2' sugar moiety is increased to a (2'S)-2'-methyl as with the SMDU-TP analog, we detect inhibition of SAMHD1's dNTPase activity. Our computational modeling suggests that (2'S)-2'-methyl sugar moiety clashing with the Y374 of SAMHD1. We speculate that SMDU-TP mechanism of action requires that the analog first docks in the catalytic pocket of SAMHD1 but prevents the A351-V378 helix conformational change from being completed, which is needed before hydrolysis can occur. Collectively we have identified stereoselective 2' substitutions that reveal nucleotide substrate specificity for SAMHD1, and a novel inhibitory mechanism for the dNTPase activity of SAMHD1. Importantly, our data is beneficial for understanding if FDA-approved antiviral and anticancer nucleosides are hydrolyzed by SAMHD1 in vivo.