To examine the role of the mitochondrial polymerase (Pol ␥) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol ␥ for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-␤-D-arabinofuranosyl)-5-iodouracil (FIAU), ␤-L-(؊)-2,3-dideoxy-3-thiacytidine (؊)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol ␥ holoenzyme in single turnover kinetic studies to measure K d (K m ) and k pol (k cat ) to estimate the specificity constant (k cat /K m ) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2,3-didehydro-2,3-dideoxythymidine (d4T) > > (؉)3TC > > (؊)3TC > PMPA > azidothymidine (AZT) > > Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s ؊1 for the series FIAU > (؉)3TC ϳ (؊)3TC > CBV > AZT > PMPA ϳ d4T > > ddA (ddI) > > ddC. Removal of ddC was too slow to measure (<0.00002 s ؊1 ). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (؊)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s ؊1 ) faster than it is removed (0.06 s ؊1 ) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.Current treatment of HIV 1 includes a mixture that generally consists of a combination of nucleoside and nonnucleoside analogs directed against HIV RT, plus an inhibitor of HIV protease. Treatment with this mixture allows patients to coexist with a low level of virus for years, but treatments are limited by the development of resistance of HIV to the drugs on the one hand and toxicity of nucleoside analogs on the other. Toxicity of nucleoside analogs is particularly troublesome for the long term management of the viral infection. Nucleoside analogs function as chain terminators to suppress viral replication by HIV-1 RT, and because HIV RT lacks a proofreading exonuclease, the specificity of nucleoside analogs toward HIV RT results from selective discrimination during incorporation and/or from removal by the proofreading exonuclease of the host DNA polymerase.Six nucleoside analogs have received Food and Drug Administration approval for treatment of HIV, and these analogs are illustrated with others...

Incorporation of nucleoside analogues by the mitochondrial DNA polymerase has been implicated as the primary cause underlying many of the toxic side effects of these drugs in HIV therapy. Recent success in reconstituting recombinant human enzyme has afforded a detailed mechanistic analysis of the reactions governing nucleotide selectivity of the polymerase and the proofreading exonuclease. The toxic side effects of nucleoside analogues are correlated with the kinetics of incorporation by the mitochondrial DNA polymerase, varying over 6 orders of magnitude in the sequence zalcitabine (ddC) > didanosine (ddI metabolized to ddA) > stavudine (d4T) >> lamivudine (3TC) > tenofovir (PMPA) > zidovudine (AZT) > abacavir (metabolized to carbovir, CBV). In this review, we summarize our current efforts to examine the mechanistic basis for nucleotide selectivity by the mitochondrial DNA polymerase and its role in mitochondrial toxicity of nucleoside analogues used to treat AIDS and other viral infections. We will also discuss the promise and underlying challenges for the development of new analogues with lower toxicity.

To assess the role of oxidative stress on the replication of mitochondrial DNA, we examined the kinetics of incorporation of 8-oxo-7,8-dihydroguanosine (8-oxodG) triphosphate catalyzed by the human mitochondrial DNA polymerase. Using transient state kinetic methods, we quantified the kinetics of incorporation, excision, and extension beyond a base pair containing 8-oxodG. The 8-oxodGTP was incorporated opposite dC in the template with a specificity constant of 0.005 M ؊1 s ؊1 , a value ϳ10,000-fold lower than that for dGTP. Once incorporated, 96% of the time 8-oxodGMP was extended by continued polymerization rather than being excised by the proofreading exonuclease. The specificity constant for incorporation of 8-oxodGTP opposite a template dA was 0.2 M ؊1 s ؊1 , a value 13-fold higher than incorporation opposite a template dC. The 8-oxodG:dA mispair was extended rather than excised at least 70% of the time. Examination of the kinetics of polymerization with 8-oxodG in the template strand also revealed relatively low fidelity in that dCTP would be incorporated only 90% of the time. In nearly 10% of events, dATP would be incorporated, and once incorporated dA (opposite 8-oxodG) was extended rather than excised. The greatest fidelity was against a dTTP:8-oxodG mismatch affording a discrimination value of only 1800. These data reveal that 8-oxodGTP is a potent mutagen. Once it is incorporated into DNA, 8-oxodGMP codes for error prone DNA synthesis. These reactions are likely to play important roles in oxidative stress in mitochondria related to aging and as compounded by nucleoside analogs used to treat human immunodeficiency virus infections.Although many theories exist regarding the underlying molecular mechanisms behind aging in mammals, it is clear that mitochondrial integrity plays a major role (1-3). According to the free radical theory of aging, electrons from the electron transport chain are able to reduce molecular oxygen to form superoxide anion radicals (O 2 . ) during aerobic respiration. These reactive radicals go on to produce other reactive oxygen species (ROS) 2 (3). ROS can be generated at a few cellular sites, but in healthy tissues the majority are a result of aerobic metabolism, and consequently, they are always present during normal cellular activity. ROS attack a variety of different cellular macromolecules, including proteins, lipids, and DNA. However, damage to mitochondrial DNA (mtDNA) has been implicated as important in regard to aging, especially in postmitotic cells, such as neurons (4). A cycle is created in the mitochondria in which a continued state of oxidative stress leads to further damage to electron transport chain components, ultimately causing an energy decline, carcinogenesis, and many age-related diseases (5).One of the most common products of oxidative DNA damage is 8-oxo-7,8-dihydroguanosine (8-oxodG), which is reported to be highly mutagenic and is commonly used as biomarker for oxidative stress. Basal levels of 8-oxodG in mtDNA between different species correlate negati...

MoaA, a radical S-adenosylmethionine (SAM) enzyme, catalyzes the first step in molybdopterin biosynthesis. This reaction involves a complex rearrangement in which C8 of guanosine triphosphtate is inserted between the C2′ and the C3′ carbons of the ribose. This study identifies the site of initial hydrogen atom abstraction by the adenosyl radical and advances a mechanistic proposal for this unprecedented reaction.

ATP-binding cassette (ABC) transporters are responsible for the transport of a wide variety of water-soluble molecules and ions into prokaryotic cells. In Gram-negative bacteria, periplasmic-binding proteins deliver ions or molecules such as thiamin to the membrane-bound ABC transporter. The gene for the thiamin-binding protein tbpA has been identified in both Escherichia coli and Salmonella typhimurium. Here we report the crystal structure of TbpA from E. coli with bound thiamin monophosphate. The structure was determined at 2.25 A resolution using single-wavelength anomalous diffraction experiments, despite the presence of nonmerohedral twinning. The crystal structure shows that TbpA belongs to the group II periplasmic-binding protein family. Equilibrium binding measurements showed similar dissociation constants for thiamin, thiamin monophosphate, and thiamin pyrophosphate. Analysis of the binding site by molecular modeling demonstrated how TbpA binds all three forms of thiamin. A comparison of TbpA and thiaminase-I, a thiamin-degrading enzyme, revealed structural similarity between the two proteins, especially in domain 1, suggesting that the two proteins evolved from a common ancestor.