The human DNA polymerase gamma (Pol c) is responsible for DNA replication in mitochondria. Pol c is particularly susceptible to inhibition by dideoxynucleoside-based inhibitors designed to fight viral infection. Here, we report crystal structures of the replicating Pol c-DNA complex bound to either substrate or zalcitabine, an inhibitor used for HIV reverse transcriptase. The structures reveal that zalcitabine binds to the Pol c active site almost identically to the substrate dCTP, providing a structural basis for Pol c-mediated drug toxicity. When compared to the apo form, Pol c undergoes intra-and inter-subunit conformational changes upon formation of the ternary complex with primer/template DNA and substrate. We also find that the accessory subunit Pol cB, which lacks intrinsic enzymatic activity and does not contact the primer/template DNA directly, serves as an allosteric regulator of holoenzyme activities. The structures presented here suggest a mechanism for processivity of the holoenzyme and provide a model for understanding the deleterious effects of Pol c mutations in human disease. Crystal structures of the mitochondrial DNA polymerase, Pol c, in complex with substrate or antiviral inhibitor zalcitabine provide a basis for understanding Pol c-mediated drug toxicity.

Pol γ, the only DNA repair and replication. During mtDNA base-excision repair, gaps are created after damaged base excision. Here we show that Pol γ efficiently gap-fills except when the gap is only a single nucleotide. Although wild-type Pol γ has very limited ability for strand displacement DNA synthesis, exo− (3′–5′ exonuclease-deficient) Pol γ has significantly high activity and rapidly unwinds downstream DNA, synthesizing DNA at a rate comparable to that of the wild-type enzyme on a primer-template. The catalytic subunit Pol γA alone, even when exo−, is unable to synthesize by strand displacement, making this the only known reaction of Pol γ holoenzyme that has an absolute requirement for the accessory subunit Pol γB.

Nucleoside analog reverse transcriptase inhibitors (NRTIs) are the essential components of highly active antiretroviral (HAART) therapy targeting HIV reverse transcriptase (RT). NRTI triphosphates (NRTI-TP), the biologically active forms, act as chain terminators of viral DNA synthesis. Unfortunately, NRTIs also inhibit human mitochondrial DNA polymerase (Pol γ), causing unwanted mitochondrial toxicity. Understanding the structural and mechanistic differences between Pol γ and RT in response to NRTIs will provide invaluable insight to aid in designing more effective drugs with lower toxicity. The NRTIs emtricitabine [(-)-2,3′-dideoxy-5-fluoro-3′-thiacytidine, (-)-FTC] and lamivudine, [(-)-2,3′-dideoxy-3′-thiacytidine, (-)-3TC] are both potent RT inhibitors, but Pol γ discriminates against (-)-FTC-TP by two orders of magnitude better than (-)-3TC-TP. Furthermore, although (-)-FTC-TP is only slightly more potent against HIV RT than its enantiomer (+)-FTC-TP, it is discriminated by human Pol γ four orders of magnitude more efficiently than (+)-FTC-TP. As a result, (-)-FTC is a much less toxic NRTI. Here, we present the structural and kinetic basis for this striking difference by identifying the discriminator residues of drug selectivity in both viral and human enzymes responsible for substrate selection and inhibitor specificity. For the first time, to our knowledge, this work illuminates the mechanism of (-)-FTC-TP differential selectivity and provides a structural scaffold for development of novel NRTIs with lower toxicity.

CXCL8/interleukin-8 is a pro-inflammatory chemokine that triggers pleiotropic responses, including inflammation, angiogenesis, wound healing and tumorigenesis. We engineered the first selective CXCR1 agonists on the basis of residue substitutions in the conserved ELR triad and CXC motif of CXCL8. Our data reveal that the molecular mechanisms of activation of CXCR1 and CXCR2 are distinct: the N-loop of CXCL8 is the major determinant for CXCR1 activation, whereas the N-terminus of CXCL8 (ELR and CXC) is essential for CXCR2 activation. We also found that activation of CXCR1 cross-desensitized CXCR2 responses in human neutrophils co-expressing both receptors, indicating that these novel CXCR1 agonists represent a new class of anti-inflammatory agents. Further, these selective CXCR1 agonists will aid at elucidating the functional significance of CXCR1 in vivo under pathophysiological conditions.

dWe found a heterozygous C2857T mutation (R953C) in polymerase gamma (Pol-␥) in an HIV-infected patient with mitochondrial toxicity. The R953C Pol-␥ 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.A ntiretroviral therapy (ART)-related toxicities predominantly manifest in mitochondrial dysfunction. A critical backbone of ART is nucleoside reverse transcriptase inhibitors (NRTIs). With widespread use of NRTIs, clinical manifestations such as lactic acidosis, lipodystrophy, peripheral neuropathies, cardiomyopathies, and pancytopenia were observed (1-3). These adverse effects of NRTIs were attributed to inhibition of the polymerase gamma enzyme (Pol-␥), responsible for mitochondrial DNA (mtDNA) replication (4). The role of mutant Pol-␥ variants in ART-related toxicity has not been systematically investigated. Only two Pol-␥ mutations (R964C and E1143G) have been associated with ART-induced mitochondrial toxicity (5, 6).We hypothesized that Pol-␥ mutations might predispose patients toward developing mitochondrial toxicity. We performed a retrospective analysis of data and specimens collected during a prospective, case-control study of ART-induced mitochondrial toxicity (i) to investigate whether Pol-␥ mutations are associated with ART-induced mitochondrial toxicity and (ii) to characterize the biochemical effect of these mutations, if any, on Pol-␥ activity. The details of the study design have been previously published (7,8). In brief, the cases comprised HIV-infected individuals identified by their HIV care providers as having symptoms consistent with ART-induced mitochondrial toxicity (2, 9). The study protocol was approved by the Institutional Review Board of the Yale School of Medicine. All participants gave their written informed consent before participation in the study.The study included 45 African Americans (15 HIV-infected individuals with mitochondrial toxicity [9], cases; 15 HIV-infected individuals without toxicity, positive controls; and 15 HIVuninfected individuals, negative controls). The demographic and clinical characteristics of participants are illustrated in Table S1 in the supplemental material. We amplified and sequenced the entire polymerase gamma (POLG) genome, comprising 22 exons, of the 45 study participants using 16 pairs of overlapping primers (see Table S2) and a previously described PCR protocol (10). We observed a heterozygous C2857T mutation in exon 18 (see Fig. S1 in the supplemental material) of the POLG catalytic active site, corresponding to a substitution of R953 in the wild type (WT) to cysteine, yielding mutant R953C (Fig. 1A), in one HIV-infected patient with mitochondrial toxicity and observed no mutations in the two control groups. The...