Lack of sugar discrimination by human Pol   requires a single glycine residue

Ruiz, José F.; Juárez, Raquel; García‐Díaz, Miguel; Terrados, Gloria; Picher, Angel J.; González-Barrera, Sergio; Henestrosa, Antonio R. Fernández de; Blanco, Luis

doi:10.1093/nar/gkg637

Cited by 91 publications

(135 citation statements)

References 31 publications

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“…6 Thus, the duplex hybrid nucleic acid would be structurally altered, having both A-and B-like qualities (48), relative to that normally encountered by pol ␤ and thereby interfering with proper binding. Similarly, catalytic efficiency of pol was severely diminished on substrates containing either a complete RNA primer or template (27,49). Consequently, although one or two ribonucleotide(s) in the upstream primer is(are) tolerated by X-family DNA polymerases, a stretch of ribonucleotides is expected to interfere with nucleic acid binding and DNA synthesis.…”

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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Section: Journal Of Biological Chemistrymentioning

confidence: 99%

DNA Polymerase β Ribonucleotide Discrimination

Cavanaugh

Beard

Wilson

2010

Journal of Biological Chemistry

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“…Early studies (reviewed in (1,2)) revealed that DNA polymerases do incorporate rNMPs during DNA synthesis. Kinetic studies (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) have further demonstrated that selectivity for insertion of dNMPs into DNA rather than rNMPs varies from 10-fold to >10 6 -fold, depending on the DNA polymerase and the dNTP/rNTP pair examined. rNMP incorporation during DNA synthesis is potentially made more probable by the fact that the concentrations of rNTPs in vivo are higher than are the concentrations of dNTPs (e.g., see refs.…”

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confidence: 99%

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

McElhinny

Watts

Kumar

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

376

582

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Measurements of nucleoside triphosphate levels in Saccharomyces cerevisiae reveal that the four rNTPs are in 36-to 190-fold molar excess over their corresponding dNTPs. During DNA synthesis in vitro using the physiological nucleoside triphosphate concentrations, yeast DNA polymerase ε, which is implicated in leading strand replication, incorporates one rNMP for every 1,250 dNMPs. Pol δ and Pol α, which conduct lagging strand replication, incorporate one rNMP for every 5,000 or 625 dNMPs, respectively. Discrimination against rNMP incorporation varies widely, in some cases by more than 100-fold, depending on the identity of the base and the template sequence context in which it is located. Given estimates of the amount of replication catalyzed by Pols α, δ, and ε, the results are consistent with the possibility that more than 10,000 rNMPs may be incorporated into the nuclear genome during each round of replication in yeast. Thus, rNMPs may be the most common noncanonical nucleotides introduced into the eukaryotic genome. Potential beneficial and negative consequences of abundant ribonucleotide incorporation into DNA are discussed, including the possibility that unrepaired rNMPs in DNA could be problematic because yeast DNA polymerase ε has difficulty bypassing a single rNMP present within a DNA template.DNA replication | nucleotide precursors | nucleotide selectivity T he integrity of the eukaryotic genome is ensured in part by the chemical nature of the storage medium-DNA. Compared to RNA, DNA is inherently more resistant to strand cleavage due to the absence of a reactive 2′ hydroxyl on the ribose ring. The active sites of most DNA polymerases are evolved to efficiently exclude ribonucleoside triphosphates (rNTPs) from being incorporated during DNA synthesis (reviewed in (1)). However, rNTP exclusion is not absolute. Early studies (reviewed in (1, 2)) revealed that DNA polymerases do incorporate rNMPs during DNA synthesis. Kinetic studies (3-13) have further demonstrated that selectivity for insertion of dNMPs into DNA rather than rNMPs varies from 10-fold to >10 6 -fold, depending on the DNA polymerase and the dNTP/rNTP pair examined. rNMP incorporation during DNA synthesis is potentially made more probable by the fact that the concentrations of rNTPs in vivo are higher than are the concentrations of dNTPs (e.g., see refs. 2, 14 and results of this study). Thus some rNMPs are likely to be stably incorporated into DNA during replication, and possibly during DNA repair, e.g., nonhomologous end joining (NHEJ) of double strand breaks in DNA (9, 15). This possibility is supported by biochemical studies implicating RNase H2 and FEN1 in the repair of single ribonucleotides in DNA (16,17). It is therefore of interest to know just how frequently rNMPs are incorporated into DNA by the DNA polymerases that synthesize the most DNA in a eukaryotic cell, namely DNA polymerases α, δ, and ε. Here we investigate this by first measuring the rNTP and dNTP concentrations in budding yeast. We then use these concentrations in DNA sy...

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“…However, pol differs from pol and pol ␤ in that it lacks dRP lyase activity (10) but relatively efficiently incorporates rNTPs into DNA (15,16). Recent evidence suggests that pol , but not pol , can fill short gaps in DNA during NHEJ even when 3Ј termini lack one or two complementary bases in the template strand.…”

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confidence: 99%

Biochemical Properties of Saccharomyces cerevisiae DNA Polymerase IV

Bębenek

García‐Díaz

Patishall

et al. 2005

Journal of Biological Chemistry

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Although mammals encode multiple family X DNA polymerases implicated in DNA repair, Saccharomyces cerevisiae has only one, DNA polymerase IV (pol IV). To better understand the repair functions of pol IV, here we characterize its biochemical properties. Like mammalian pol ␤ and pol , but not pol , pol IV has intrinsic 5-2-deoxyribose-5-phosphate lyase activity. Pol IV has low processivity and can fill short gaps in DNA. Unlike the case with pol ␤ and pol , the gap-filling activity of pol IV is not enhanced by a 5-phosphate on the downstream primer but is stimulated by a 5-terminal synthetic abasic site. Pol IV incorporates rNTPs into DNA with an unusually high efficiency relative to dNTPs, a property in common with pol but not pol ␤ or pol . Finally, pol IV is highly inaccurate, with an unusual error specificity indicating the ability to extend primer termini with limited homology. These properties are consistent with a possible role for pol IV in base excision repair and with its known role in non-homologous end joining of double strand breaks, perhaps including those with damaged ends.Family X DNA polymerases are evolutionarily conserved, single-subunit enzymes devoid of 3Ј 3 5Ј exonuclease activity. The most extensively studied family X polymerase (pol) 1 is mammalian DNA pol ␤, which has a major role in base excision repair (1). Other mammalian family X polymerases also implicated in DNA repair include template-dependent pol (2) and pol (3-5) and template-independent terminal deoxyribonucleotidyl transferase (TdT) (6). These enzymes share an 8-kDa domain and a polymerase domain comprised of fingers, palm, and thumb subdomains. Nonetheless, they differ in several amino acids likely to be important for function. Pol , pol , and TdT also have N-terminal BRCT domains not present in pol ␤. Mammalian family X polymerases also have different biochemical properties. TdT adds nucleotides in a template-independent manner, consistent with its role in increasing the diversity of antigen receptor genes by adding nucleotides to the ends of coding segments during V(D)J recombination (7). Pol ␤ is template-dependent and moderately accurate for both substitution and single base deletion errors; it fills short gaps in DNA in a manner stimulated by a 5Ј-phosphate on the downstream primer (8) and has an intrinsic dRP lyase activity (9). These properties are all consistent with the well established role of pol ␤ in BER.Pol shares most of the above-mentioned properties with pol ␤, suggesting that pol may also function in BER (10, 11). Pol binds dNTPs with high affinity, consistent with polymerization when cellular dNTP concentrations are low, e.g. in cells not in S phase (12). Pol generates single base deletions, especially in non-iterated sequence contexts, at a much higher rate than pol ␤, indicating an ability to use template-primers with minimal base pairing homology. These properties, in combination with a BRCT domain and the ability to physically and functionally interact with several proteins involved in NHEJ of double st...

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Lack of sugar discrimination by human Pol requires a single glycine residue

Cited by 91 publications

References 31 publications

DNA Polymerase β Ribonucleotide Discrimination

DNA Polymerase β Ribonucleotide Discrimination

Abundant ribonucleotide incorporation into DNA by yeast replicative polymerases

Biochemical Properties of Saccharomyces cerevisiae DNA Polymerase IV

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