An interaction network in the polymerase active site is a prerequisite for Watson-Crick base pairing in Pol γ.

The replication accuracy of DNA polymerase gamma (Pol γ) is essential for mitochondrial genome integrity. Mutation of human Pol γ arginine-853 has been linked to neurological diseases. Although not a catalytic residue, Pol γ arginine-853 mutants are void of polymerase activity. To identify the structural basis for the disease, we determined a crystal structure of the Pol γ mutant ternary complex with correct incoming nucleotide 2'-deoxycytidine 5'-triphosphate (dCTP). Opposite to the wild type that undergoes open-to-closed conformational changes when bound to a correct nucleotide that is essential for forming a catalytically competent active site, the mutant complex failed to undergo the conformational change, and the dCTP did not base pair with its Watson-Crick complementary templating residue. Our studies revealed that arginine-853 coordinates an interaction network that aligns the 3'-end of primer and dCTP with the catalytic residues. Disruption of the network precludes the formation of Watson-Crick base pairing and closing of the active site, resulting in an inactive polymerase.

Decoration of Burkholderia Hcp1 protein to virus-like particles as a vaccine delivery platform.

Virus-like particles (VLPs) are protein-based nanoparticles frequently used as carriers in conjugate vaccine platforms. VLPs have been used to display foreign antigens for vaccination and to deliver immunotherapy against diseases. Hemolysin-coregulated proteins 1 (Hcp1) is a protein component of the Burkholderia type 6 secretion system, which participates in intracellular invasion and dissemination. This protein has been reported as a protective antigen and is used in multiple vaccine candidates with various platforms against melioidosis, a severe infectious disease caused by the intracellular pathogen Burkholderia pseudomallei. In this study, we used P22 VLPs as a surface platform for decoration with Hcp1 using chemical conjugation. C57BL/6 mice were intranasally immunized with three doses of either PBS, VLPs, or conjugated Hcp1-VLPs. Immunization with Hcp1-VLPs formulation induced Hcp1-specific IgG, IgG1, IgG2c, and IgA antibody responses. Furthermore, the serum from Hcp1-VLPs immunized mice enhanced the bacterial uptake and opsonophagocytosis by macrophages in the presence of complement. This study demonstrated an alternative strategy to develop a VLPs-based vaccine platform against Burkholderia species.

The Role of Poly(ADP-ribose) Polymerase 1 in Nuclear and Mitochondrial Base Excision Repair.

Poly(ADP-ribose) (PAR) Polymerase 1 (PARP-1), also known as ADP-ribosyl transferase with diphtheria toxin homology 1 (ARTD-1), is a critical player in DNA damage repair, during which it catalyzes the ADP ribosylation of self and target enzymes. While the nuclear localization of PARP-1 has been well established, recent studies also suggest its mitochondrial localization. In this review, we summarize the differences between mitochondrial and nuclear Base Excision Repair (BER) pathways, the involvement of PARP-1 in mitochondrial and nuclear BER, and its functional interplay with other BER enzymes.

Maintenance of Flap Endonucleases for Long-Patch Base Excision DNA Repair in Mouse Muscle and Neuronal Cells Differentiated In Vitro.

After cellular differentiation, nuclear DNA is no longer replicated, and many of the associated proteins are downregulated accordingly. These include the structure-specific endonucleases Fen1 and DNA2, which are implicated in repairing mitochondrial DNA (mtDNA). Two more such endonucleases, named MGME1 and ExoG, have been discovered in mitochondria. This category of nuclease is required for so-called "long-patch" (multinucleotide) base excision DNA repair (BER), which is necessary to process certain oxidative lesions, prompting the question of how differentiation affects the availability and use of these enzymes in mitochondria. In this study, we demonstrate that Fen1 and DNA2 are indeed strongly downregulated after differentiation of neuronal precursors (Cath.a-differentiated cells) or mouse myotubes, while the expression levels of MGME1 and ExoG showed minimal changes. The total flap excision activity in mitochondrial extracts of these cells was moderately decreased upon differentiation, with MGME1 as the predominant flap endonuclease and ExoG playing a lesser role. Unexpectedly, both differentiated cell types appeared to accumulate less oxidative or alkylation damage in mtDNA than did their proliferating progenitors. Finally, the overall rate of mtDNA repair was not significantly different between proliferating and differentiated cells. Taken together, these results indicate that neuronal cells maintain mtDNA repair upon differentiation, evidently relying on mitochondria-specific enzymes for long-patch BER.

A highly selective humanized DDR1 mAb reverses immune exclusion by disrupting collagen fiber alignment in breast cancer.

BACKGROUND: Immune exclusion (IE) where tumors deter the infiltration of immune cells into the tumor microenvironment has emerged as a key mechanism underlying immunotherapy resistance. We recently reported a novel role of discoidin domain-containing receptor 1 (DDR1) in promoting IE in breast cancer and validated its critical role in IE using neutralizing rabbit monoclonal antibodies (mAbs) in multiple mouse tumor models. METHODS: To develop a DDR1-targeting mAb as a potential cancer therapeutic, we humanized mAb9 with a complementarity-determining region grafting strategy. The humanized antibody named PRTH-101 is currently being tested in a Phase 1 clinical trial. We determined the binding epitope of PRTH-101 from the crystal structure of the complex between DDR1 extracellular domain (ECD) and the PRTH-101 Fab fragment with 3.15 Å resolution. We revealed the underlying mechanisms of action of PRTH-101 using both cell culture assays and in vivo study in a mouse tumor model. RESULTS: PRTH-101 has subnanomolar affinity to DDR1 and potent antitumor efficacy similar to the parental rabbit mAb after humanization. Structural information illustrated that PRTH-101 interacts with the discoidin (DS)-like domain, but not the collagen-binding DS domain of DDR1. Mechanistically, we showed that PRTH-101 inhibited DDR1 phosphorylation, decreased collagen-mediated cell attachment, and significantly blocked DDR1 shedding from the cell surface. Treatment of tumor-bearing mice with PRTH-101 in vivo disrupted collagen fiber alignment (a physical barrier) in the tumor extracellular matrix (ECM) and enhanced CD8+ T cell infiltration in tumors. CONCLUSIONS: This study not only paves a pathway for the development of PRTH-101 as a cancer therapeutic, but also sheds light on a new therapeutic strategy to modulate collagen alignment in the tumor ECM for enhancing antitumor immunity.

Polγ coordinates DNA synthesis and proofreading to ensure mitochondrial genome integrity.

Accurate replication of mitochondrial DNA (mtDNA) by DNA polymerase γ (Polγ) is essential for maintaining cellular energy supplies, metabolism, and cell cycle control. To illustrate the structural mechanism for Polγ coordinating polymerase (pol) and exonuclease (exo) activities to ensure rapid and accurate DNA synthesis, we determined four cryo-EM structures of Polγ captured after accurate or erroneous incorporation to a resolution of 2.4-3.0 Å. The structures show that Polγ employs a dual-checkpoint mechanism to sense nucleotide misincorporation and initiate proofreading. The transition from replication to error editing is accompanied by increased dynamics in both DNA and enzyme, in which the polymerase relaxes its processivity and the primer-template DNA unwinds, rotates, and backtracks to shuttle the mismatch-containing primer terminus 32 Å to the exo site for editing. Our structural and functional studies also provide a foundation for analyses of Polγ mutation-induced human diseases and aging.

Structural and Molecular Basis for Mitochondrial DNA Replication and Transcription in Health and Antiviral Drug Toxicity.

Human mitochondrial DNA (mtDNA) is a 16.9 kbp double-stranded, circular DNA, encoding subunits of the oxidative phosphorylation electron transfer chain and essential RNAs for mitochondrial protein translation. The minimal human mtDNA replisome is composed of the DNA helicase Twinkle, DNA polymerase γ, and mitochondrial single-stranded DNA-binding protein. While the mitochondrial RNA transcription is carried out by mitochondrial RNA polymerase, mitochondrial transcription factors TFAM and TFB2M, and a transcription elongation factor, TEFM, both RNA transcriptions, and DNA replication machineries are intertwined and control mtDNA copy numbers, cellular energy supplies, and cellular metabolism. In this review, we discuss the mechanisms governing these main pathways and the mtDNA diseases that arise from mutations in transcription and replication machineries from a structural point of view. We also address the adverse effect of antiviral drugs mediated by mitochondrial DNA and RNA polymerases as well as possible structural approaches to develop nucleoside reverse transcriptase inhibitor and ribonucleosides analogs with reduced toxicity.

Human EXOG Possesses Strong AP Hydrolysis Activity: Implication on Mitochondrial DNA Base Excision Repair.

Most oxidative damage on mitochondrial DNA is corrected by the base excision repair (BER) pathway. However, the enzyme that catalyzes the rate-limiting reaction─deoxyribose phosphate (dRP) removal─in the multienzymatic reaction pathway has not been completely determined in mitochondria. Also unclear is how a logical order of enzymatic reactions is ensured. Here, we present structural and enzymatic studies showing that human mitochondrial EXOG (hEXOG) exhibits strong 5'-dRP removal ability. We show that, unlike the canonical dRP lyases that act on a single substrate, hEXOG functions on a variety of abasic sites, including 5'-dRP, its oxidized product deoxyribonolactone (dL), and the stable synthetic analogue tetrahydrofuran (THF). We determined crystal structures of hEXOG complexed with a THF-containing DNA and with a partial gapped DNA to 2.9 and 2.1 Šresolutions, respectively. The structures illustrate that hEXOG uses a controlled 5'-exonuclease activity to cleave the third phosphodiester bond away from the 5'-abasic site. This study provides a structural basis for hEXOG's broad spectrum of substrates. Further, we show that hEXOG can set the order of BER reactions by generating an ideal substrate for the subsequent reaction in BER and inhibit off-pathway reactions.

Human Mitochondrial DNA Polymerase Metal Dependent UV Lesion Bypassing Ability.

Human mitochondrial DNA contains more UV-induced lesions than the nuclear DNA due to lack of mechanism to remove bulky photoproducts. Human DNA polymerase gamma (Pol γ) is the sole DNA replicase in mitochondria, which contains a polymerase (pol) and an exonuclease (exo) active site. Previous studies showed that Pol γ only displays UV lesion bypassing when its exonuclease activity is obliterated. To investigate the reaction environment on Pol γ translesion activity, we tested Pol γ DNA activity in the presence of different metal ions. While Pol γ is unable to replicate through UV lesions on DNA templates in the presence of Mg2+, it exhibits robust translesion DNA synthesis (TLS) on cyclobutane pyrimidine dimer (CPD)-containing template when Mg2+ was mixed with or completely replaced by Mn2+. Under these conditions, the efficiency of Pol γ's TLS opposite CPD is near to that on a non-damaged template and is 800-fold higher than that of exonuclease-deficient Pol γ. Interestingly, Pol γ exhibits higher exonuclease activity in the presence of Mn2+ than with Mg2+, suggesting Mn2+-stimulated Pol γ TLS is not via suppressing its exonuclease activity. We suggest that Mn2+ ion expands Pol γ's pol active site relative to Mg2+ so that a UV lesion can be accommodated and blocks the communication between pol and exo active sites to execute translesion DNA synthesis.

Cell-Type Apoptosis in Lung during SARS-CoV-2 Infection.

The SARS-CoV-2 pandemic has inspired renewed interest in understanding the fundamental pathology of acute respiratory distress syndrome (ARDS) following infection. However, the pathogenesis of ARDS following SRAS-CoV-2 infection remains largely unknown. In the present study, we examined apoptosis in postmortem lung sections from COVID-19 patients and in lung tissues from a non-human primate model of SARS-CoV-2 infection, in a cell-type manner, including type 1 and 2 alveolar cells and vascular endothelial cells (ECs), macrophages, and T cells. Multiple-target immunofluorescence assays and Western blotting suggest both intrinsic and extrinsic apoptotic pathways are activated during SARS-CoV-2 infection. Furthermore, we observed that SARS-CoV-2 fails to induce apoptosis in human bronchial epithelial cells (i.e., BEAS2B cells) and primary human umbilical vein endothelial cells (HUVECs), which are refractory to SARS-CoV-2 infection. However, infection of co-cultured Vero cells and HUVECs or Vero cells and BEAS2B cells with SARS-CoV-2 induced apoptosis in both Vero cells and HUVECs/BEAS2B cells but did not alter the permissiveness of HUVECs or BEAS2B cells to the virus. Post-exposure treatment of the co-culture of Vero cells and HUVECs with a novel non-cyclic nucleotide small molecule EPAC1-specific activator reduced apoptosis in HUVECs. These findings may help to delineate a novel insight into the pathogenesis of ARDS following SARS-CoV-2 infection.

Poly(ADP-ribose) polymerase 1 regulates mitochondrial DNA repair in an NAD-dependent manner.

Mitochondrial DNA is located in organelle that house essential metabolic reactions and contains high reactive oxygen species. Therefore, mitochondrial DNA suffers more oxidative damage than its nuclear counterpart. Formation of a repair enzyme complex is beneficial to DNA repair. Recent studies have shown that mitochondrial DNA polymerase (Pol γ) and poly(ADP-ribose) polymerase 1 (PARP1) were found in the same complex along with other mitochondrial DNA repair enzymes, and mitochondrial PARP1 level is correlated with mtDNA integrity. However, the molecular basis for the functional connection between Pol γ and PARP1 has not yet been elucidated because cellular functions of PARP1 in DNA repair are intertwined with metabolism via NAD+ (nicotinamide adenosine dinucleotide), the substrate of PARP1, and a metabolic cofactor. To dissect the direct effect of PARP1 on mtDNA from the secondary perturbation of metabolism, we report here biochemical studies that recapitulated Pol γ PARylation observed in cells and showed that PARP1 regulates Pol γ activity during DNA repair in a metabolic cofactor NAD+ (nicotinamide adenosine dinucleotide)-dependent manner. In the absence of NAD+, PARP1 completely inhibits Pol γ, while increasing NAD+ levels to a physiological concentration that enables Pol γ to resume maximum repair activity. Because cellular NAD+ levels are linked to metabolism and to ATP production via oxidative phosphorylation, our results suggest that mtDNA damage repair is coupled to cellular metabolic state and the integrity of the respiratory chain.

Transcription polymerase-catalyzed emergence of novel RNA replicons.

Transcription polymerases can exhibit an unusual mode of regenerating certain RNA templates from RNA, yielding systems that can replicate and evolve with RNA as the information carrier. Two classes of pathogenic RNAs (hepatitis delta virus in animals and viroids in plants) are copied by host transcription polymerases. Using in vitro RNA replication by the transcription polymerase of T7 bacteriophage as an experimental model, we identify hundreds of new replicating RNAs, define three mechanistic hallmarks of replication (subterminal de novo initiation, RNA shape-shifting, and interrupted rolling-circle synthesis), and describe emergence from DNA seeds as a mechanism for the origin of novel RNA replicons. These results inform models for the origins and replication of naturally occurring RNA genetic elements and suggest a means by which diverse RNA populations could be propagated as hereditary material in cellular contexts.

Excessive excision of correct nucleotides during DNA synthesis explained by replication hurdles.

The proofreading exonuclease activity of replicative DNA polymerase excises misincorporated nucleotides during DNA synthesis, but these events are rare. Therefore, we were surprised to find that T7 replisome excised nearly 7% of correctly incorporated nucleotides during leading and lagging strand syntheses. Similar observations with two other DNA polymerases establish its generality. We show that excessive excision of correctly incorporated nucleotides is not due to events such as processive degradation of nascent DNA or spontaneous partitioning of primer-end to the exonuclease site as a "cost of proofreading". Instead, we show that replication hurdles, including secondary structures in template, slowed helicase, or uncoupled helicase-polymerase, increase DNA reannealing and polymerase backtracking, and generate frayed primer-ends that are shuttled to the exonuclease site and excised efficiently. Our studies indicate that active-site shuttling occurs at a high frequency, and we propose that it serves as a proofreading mechanism to protect primer-ends from mutagenic extensions.

Structural Energy Landscapes and Plasticity of the Microstates of Apo Escherichia coli cAMP Receptor Protein.

The theory for allostery has evolved to a modern energy landscape ensemble theory, the major feature of which is the existence of multiple microstates in equilibrium. The properties of microstates are not well defined due to their transient nature. Characterization of apo protein microstates is important because the specific complex of the ligand-bound microstate defines the biological function. The information needed to link biological function and structure is a quantitative correlation of the energy landscapes between the apo and holo protein states. We employed the Escherichia coli cAMP receptor protein (CRP) system to test the features embedded in the ensemble theory because multiple crystalline apo and holo structures are available. Small angle X-ray scattering data eliminated one of the three apo states but not the other two. We defined the underlying energy landscape differences among the apo microstates by employing the computation algorithm COREX/BEST. The same connectivity patterns among residues in apo CRP are retained upon binding of cAMP. The microstates of apo CRP differ from one another by minor structural perturbations, resulting in changes in the energy landscapes of the various domains of CRP. Using the differences in energy landscapes among these apo states, we computed the cAMP binding energetics that were compared with solution biophysical results. Only one of the three apo microstates yielded data consistent with the solution data. The relative magnitude of changes in energy landscapes embedded in various apo microstates apparently defines the ultimate outcome of the cooperativity of binding.

Oxidative damage diminishes mitochondrial DNA polymerase replication fidelity.

Mitochondrial DNA (mtDNA) resides in a high ROS environment and suffers more mutations than its nuclear counterpart. Increasing evidence suggests that mtDNA mutations are not the results of direct oxidative damage, rather are caused, at least in part, by DNA replication errors. To understand how the mtDNA replicase, Pol γ, can give rise to elevated mutations, we studied the effect of oxidation of Pol γ on replication errors. Pol γ is a high fidelity polymerase with polymerase (pol) and proofreading exonuclease (exo) activities. We show that Pol γ exo domain is far more sensitive to oxidation than pol; under oxidative conditions, exonuclease activity therefore declines more rapidly than polymerase. The oxidized Pol γ becomes editing-deficient, displaying a 20-fold elevated mutations than the unoxidized enzyme. Mass spectrometry analysis reveals that Pol γ exo domain is a hotspot for oxidation. The oxidized exo residues increase the net negative charge around the active site that should reduce the affinity to mismatched primer/template DNA. Our results suggest that the oxidative stress induced high mutation frequency on mtDNA can be indirectly caused by oxidation of the mitochondrial replicase.

Structural insights into the recognition of nucleoside reverse transcriptase inhibitors by HIV-1 reverse transcriptase: First crystal structures with reverse transcriptase and the active triphosphate forms of lamivudine and emtricitabine.

The retrovirus HIV-1 has been a major health issue since its discovery in the early 80s. In 2017, over 37 million people were infected with HIV-1, of which 1.8 million were new infections that year. Currently, the most successful treatment regimen is the highly active antiretroviral therapy (HAART), which consists of a combination of three to four of the current 26 FDA-approved HIV-1 drugs. Half of these drugs target the reverse transcriptase (RT) enzyme that is essential for viral replication. One class of RT inhibitors is nucleoside reverse transcriptase inhibitors (NRTIs), a crucial component of the HAART. Once incorporated into DNA, NRTIs function as a chain terminator to stop viral DNA replication. Unfortunately, treatment with NRTIs is sometimes linked to toxicity caused by off-target side effects. NRTIs may also target the replicative human mitochondrial DNA polymerase (Pol γ), causing long-term severe drug toxicity. The goal of this work is to understand the discrimination mechanism of different NRTI analogues by RT. Crystal structures and kinetic experiments are essential for the rational design of new molecules that are able to bind selectively to RT and not Pol γ. Structural comparison of NRTI-binding modes with both RT and Pol γ enzymes highlights key amino acids that are responsible for the difference in affinity of these drugs to their targets. Therefore, the long-term goal of this research is to develop safer, next generation therapeutics that can overcome off-target toxicity.

Networked Communication between Polymerase and Exonuclease Active Sites in Human Mitochondrial DNA Polymerase.

High fidelity human mitochondrial DNA polymerase (Pol γ) contains two active sites, a DNA polymerization site (pol) and a 3'-5' exonuclease site (exo) for proofreading. Although separated by 35 Å, coordination between the pol and exo sites is crucial to high fidelity replication. The biophysical mechanisms for this coordination are not completely understood. To understand the communication between the two active sites, we used a statistical-mechanical model of the protein ensemble to calculate the energetic landscape and local stability. We compared a series of structures of Pol γ, complexed with primer/template DNA, and either a nucleotide substrate or a series of nucleotide analogues, which are differentially incorporated and excised by pol and exo activity. Despite the nucleotide or its analogues being bound in the pol, Pol γ residue stability varied across the protein, particularly in the exo domain. This suggests that substrate presence in the pol can be "sensed" in the exo domain. Consistent with this hypothesis, in silico mutations made in one active site mutually perturbed the energetics of the other. To identify specific regions of the polymerase that contributed to this communication, we constructed an allosteric network connectivity map that further demonstrates specific pol-exo cooperativity. Thus, a cooperative network underlies energetic connectivity. We propose that Pol γ and other dual-function polymerases exploit an energetic coupling network that facilitates domain-domain communication to enhance discrimination between correct and incorrect nucleotides.

A domain in human EXOG converts apoptotic endonuclease to DNA-repair exonuclease.

Human EXOG (hEXOG) is a 5'-exonuclease that is crucial for mitochondrial DNA repair; the enzyme belongs to a nonspecific nuclease family that includes the apoptotic endonuclease EndoG. Here we report biochemical and structural studies of hEXOG, including structures in its apo form and in a complex with DNA at 1.81 and 1.85 Å resolution, respectively. A Wing domain, absent in other ßßα-Me members, suppresses endonuclease activity, but confers on hEXOG a strong 5'-dsDNA exonuclease activity that precisely excises a dinucleotide using an intrinsic 'tape-measure'. The symmetrical apo hEXOG homodimer becomes asymmetrical upon binding to DNA, providing a structural basis for how substrate DNA bound to one active site allosterically regulates the activity of the other. These properties of hEXOG suggest a pathway for mitochondrial BER that provides an optimal substrate for subsequent gap-filling synthesis by DNA polymerase γ.

The DNA Polymerase Gamma R953C Mutant Is Associated with Antiretroviral Therapy-Induced Mitochondrial Toxicity.

We found a heterozygous C2857T mutation (R953C) in polymerase gamma (Pol-γ) in an HIV-infected patient with mitochondrial toxicity. The R953C Pol-γ mutant binding affinity for dCTP is 8-fold less than that of the wild type. The R953C mutant shows a 4-fold decrease in discrimination of analog nucleotides relative to the wild type. R953 is located on the "O-helix" that forms the substrate deoxynucleoside triphosphate (dNTP) binding site; the interactions of R953 with E1056 and Y986 may stabilize the O-helix and affect polymerase activity.