Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

Boyer, Paul L.; Sarafianos, Stefan G.; Arnold, Edward; Hughes, Stephen H.

doi:10.1073/pnas.97.7.3056

Cited by 58 publications

(58 citation statements)

References 32 publications

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“…On random DNA substrates, Mn 2ϩ causes substantial misincorporation, which stalls replication due to the inefficiency of the DNA polymerase to extend a mismatch (44). However, on homopolymer substrates, such as with oligo(dT) [12][13][14][15][16][17][18] /poly(rA) with only dTTP, the catalytic rate appears higher as compared with natural DNA. In vivo, DNA polymerases use Mg 2ϩ as the metal co-activator, which promotes high fidelity polymerization.…”

Section: Discussionmentioning

confidence: 99%

“…Efficient discrimination of ribonucleotides by these enzymes is controlled by specific amino acid residues, which sterically block the incoming rNMP to the enzyme's active site. However, the identity of these steric gate side chains varies between different polymerases (15)(16)(17)(18). Goff and colleagues (14) first identified a phenylalanine residue (Phe-155) in Moloney murine leukemia viral reverse transcriptase that confers selectivity against ribonucleotides.…”

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confidence: 99%

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Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase

Kasiviswanathan

Copeland

2011

Journal of Biological Chemistry

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During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared with deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from their active site through a bulky side chain residue that can sterically block the 2-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase ␥ discriminates ribonucleotides efficiently but differentially based on the base identity. Whereas UTP is discriminated by 77,000-fold compared with dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme was reduced 3-14-fold, depending on the identity of the incoming nucleotide, when it extended from a primer containing a 3-terminal ribonucleotide. DNA polymerase ␥ is also proficient in performing single-nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides, mainly due to its defects in deoxyribonucleoside triphosphate binding, and is also a poor reverse transcriptase. The potential biochemical defects of a patient harboring a disease mutation in the same amino acid (E895G) are discussed.Replicative DNA polymerases must have the ability not only to discriminate between the correct and incorrect nucleotide to be incorporated during DNA synthesis but also to distinguish between the correct and incorrect sugar moiety to maintain its fidelity. This process is crucial because ribonucleotides are more prone to strand cleavage compared with deoxyribonucleotides due to the presence of a reactive hydroxyl group at the 2Ј-position of the ribose sugar. The discrimination against ribonucleotide incorporation during various DNA metabolic processes has been studied for many nuclear replicative DNA polymerases, including pol 2 ␣, ␦, and ⑀ and other polymerases involved in DNA repair like pol ␤, , and (1-5). Studies on these eukaryotic nuclear replicative and repair enzymes indicate that the levels of ribonucleotide discrimination vary from 2 to 3 orders of magnitude between them, suggesting that the mechanism employed by each polymerase in excluding ribonucleotide from its active site may be different during DNA synthesis. Analysis of the human DNA polymerase ␤ revealed that this polymerase discriminates against ribonucleotide incorporation by nearly 4 orders of magnitude (3). DNA pol ␥, encoded by the nuclear gene POLG, is the sole DNA polymerase in human mitochondria and hence bears the burden of replicating and repairing the entire mitochondrial DNA (6, 7). The holoenzyme of pol ␥ is a heterotrimer composed of a catalytic subunit (p140) and a homodimeric accessory subunit (p55...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase

Kasiviswanathan

Copeland

2011

Journal of Biological Chemistry

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“…Although the efficiency for ribonucleotide insertion appears to be independent of polymerase identity, the strategy employed by X-family polymerases used to sterically deter ribonucleotide insertion is unique. DNA polymerases from other families employ a protein side chain to sterically exclude ribonucleotides (4,8,(31)(32)(33). In contrast, this exclusion is primarily provided by the protein backbone for X-family members (Fig.…”

Section: Journal Of Biological Chemistrymentioning

confidence: 99%

DNA Polymerase β Ribonucleotide Discrimination

Cavanaugh

Beard

Wilson

2010

Journal of Biological Chemistry

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DNA polymerases must select nucleotides that preserve Watson-Crick base pairing rules and choose substrates with the correct (deoxyribose) sugar. Sugar discrimination represents a great challenge because ribonucleotide triphosphates are present at much higher cellular concentrations than their deoxycounterparts. Although DNA polymerases discriminate against ribonucleotides, many therapeutic nucleotide analogs that target polymerases have sugar modifications, and their efficacy depends on their ability to be incorporated into DNA. Here, we investigate the ability of DNA polymerase ␤ to utilize nucleotides with modified sugars. DNA polymerase ␤ readily inserts dideoxynucleoside triphosphates but inserts ribonucleotides nearly 4 orders of magnitude less efficiently than natural deoxynucleotides. The efficiency of ribonucleotide insertion is similar to that reported for other DNA polymerases. The poor polymerase-dependent insertion represents a key step in discriminating against ribonucleotides because, once inserted, a ribonucleotide is easily extended. Likewise, a templating ribonucleotide has little effect on insertion efficiency or fidelity. In contrast to insertion and extension of a ribonucleotide, the chemotherapeutic drug arabinofuranosylcytosine triphosphate is efficiently inserted but poorly extended. These results suggest that the sugar pucker at the primer terminus plays a crucial role in DNA synthesis; a 3-endo sugar pucker facilitates nucleotide insertion, whereas a 2-endo conformation inhibits insertion.To maintain faithful DNA synthesis, DNA polymerases have evolved to select a dNTP from a pool of structurally similar molecules that preserve Watson-Crick base pairing. This is facilitated by geometric constraints (size, shape, and hydrogen bonding potential) imposed by the template strand, primer terminus, and polymerase. Although the fidelity of base substitution errors, and their correction, has been extensively studied, the fidelity of sugar discrimination has received much less attention. It is well recognized that the dNTP pool imbalances influence DNA polymerase fidelity. In this context, cellular rNTP levels are far greater than their dNTP counterparts (1, 2). To prevent significant levels of RNA synthesis during replication and repair, DNA polymerases must inherently discriminate against nucleotides with a ribose sugar (i.e. possessing a 2Ј-OH) and select 2Ј-deoxyribose triphosphates. Previous studies with A-and B-family polymerases have found significant effects on nucleotide incorporation as a result of modifying the deoxyribose ring (3-8). DNA polymerases insert ribonucleotides with a much lower efficiency than deoxynucleotides with the same base due to a slower rate of insertion and weaker binding.DNA polymerase (pol) 2 ␤, an X-family member that also includes pol , pol , and terminal deoxyribonucleotidyltransferase (TdT), has been well characterized kinetically, structurally, and biochemically (9) making it a model DNA polymerase to probe sugar specificity. DNA polymerase ␤ is a critical ...

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“…Tyr 115 is known to exclude the binding of RNA nucleotides by sterically disallowing the presence of a 2′-hydroxyl, leading it to be referred to as the steric gate of RT (Ding et al, 1998;Boyer et al, 2000). A similarly positioned residue in other polymerases is considered to have the same function ( Joyce, 1997;Astatke et al, 1998).…”

Section: Structural Implications For the Kinetic Datamentioning

confidence: 99%

Probing the Mechanistic Consequences of 5-Fluorine Substitution on Cytidine Nucleotide Analogue Incorporation by HIV-1 Reverse Transcriptase

Ray

Schinazi

Murakami

et al. 2003

Antivir Chem Chemother

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β-D and β-L-enantiomers of 2′,3′-dideoxycytidine analogues are potent chain-terminators and antimetabolites for viral and cellular replication. Seemingly small modifications markedly alter their antiviral and toxicity patterns. This review discusses previously published and recently obtained data on the effects of 5- and 2′-fluorine substitution on the pre-steady state incorporation of 2′-deoxycytidine-5′-monophosphate analogues by HIV-1 reverse transcriptase (RT) in light of their biological activity. The addition of fluorine at the 5-position of the pyrimidine ring altered the kinetic parameters for all nucleotides tested. Only the 5-fluorine substitution of the clinically relevant nucleosides (-)-β-L-2′,3′-dideoxy-3′-thia-5-fluoro-cytidine (L-FTC, Emtriva™), and (+)-β-D-2′,3′-dide-hydro-2′,3′-dideoxy-5-fluorocytidine (D-D4FC, Reverset™), caused a higher overall efficiency of nucleotide incorporation during both DNA- and RNA-directed synthesis. Enhanced incorporation by RT may in part explain the potency of these nucleosides against HIV-1. In other cases, a lack of correlation between RT incorporation in enzymatic assays and antiviral activity in cell culture illustrates the importance of other cellular factors in defining antiviral potency. The substitution of fluorine at the 2′ position of the deoxyribose ring negatively affects incorporation by RT indicating the steric gate of RT can detect electrostatic perturbations. Intriguing results pertaining to drug resistance have led to a better understanding of HIV-1 RT resistance mechanisms. These insights serve as a basis for understanding the mechanism of action for nucleoside analogues and, coupled with studies on other key enzymes, may lead to the more effective use of fluorine to enhance the potency and selectivity of antiviral agents.

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Analysis of mutations at positions 115 and 116 in the dNTP binding site of HIV-1 reverse transcriptase

Cited by 58 publications

References 32 publications

Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase

Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase

DNA Polymerase β Ribonucleotide Discrimination

Probing the Mechanistic Consequences of 5-Fluorine Substitution on Cytidine Nucleotide Analogue Incorporation by HIV-1 Reverse Transcriptase

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