Base Substitution Specificity of DNA Polymerase β Depends on Interactions in the DNA Minor Groove

Osheroff, Wendy P.; Beard, William A.; Wilson, Samuel H.; Kunkel, Thomas A.

doi:10.1074/jbc.274.30.20749

Cited by 66 publications

(69 citation statements)

References 31 publications

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“…The results in Table 1 illustrate that a single amino acid difference not only can reduce replication fidelity but can do so in a highly selective manner, affecting some substitutions but not others. In this regard, the E710A mutant Klenow polymerase is similar to the R283K mutant of DNA polymerase ␤, which has elevated error rates for some mismatches and wild-type rates for others (27). In both instances, these selective influences on error rates are inferred to reflect altered interactions in the binding pocket for the nascent base pair that involve the mutated amino acid side chains and the DNA minor groove.…”

Section: Discussionmentioning

confidence: 97%

Discrimination against purine–pyrimidine mispairs in the polymerase active site of DNA polymerase I: A structural explanation

Minnick

Liu

Grindley

et al. 2002

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

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We previously identified five derivatives of Klenow fragment DNA polymerase that have lower fidelity because of amino acid substitutions in the polymerase active site. One of these has alanine substituted for the invariant Glu-710, whose side chain interacts with the deoxyribose of the incoming dNTP. Here we show that the E710A enzyme has reduced fidelity for five of the 12 possible mismatches. All but one of these involve misinsertion of pyrimidines, including two transition mismatches A-dCTP and G-dTTP. In contrast, E710A polymerase error rates for the reciprocal C-dATP and T-dGTP transition mismatches were similar to those of the wild-type enzyme. The kinetics of formation of correct base pairs and transition mismatches by the wild-type and E710A polymerases, combined with information on the structure of the DNA polymerase active site and the asymmetry of wobble base pairs, provides a plausible explanation for the differential effects of the E710A mutation on fidelity. The data suggest that the Glu-710 side chain plays a pivotal role in excluding wobble base pairs between template pyrimidines and purine triphosphates by steric clash. Moreover, this same side chain enhances the stability of incoming correct dNTPs, such that loss of this interaction on removal of the side chain leads to lower selectivity against mismatches involving incoming pyrimidines.

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Section: Discussionmentioning

confidence: 97%

Discrimination against purine–pyrimidine mispairs in the polymerase active site of DNA polymerase I: A structural explanation

Minnick

Liu

Grindley

et al. 2002

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

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“…In addition, a recently solved ternary complex structure of Sulfolobus solfataricus P2 DNA polymerase (Dpo4), which exhibits a very low fidelity, revealed that the nascent template-primer DNA is in the B-form (42,43). Based on structural and biochemical studies, a common fidelity mechanism involving hydrogen-bonding interactions between the polymerase and the minor groove of the template-primer has been proposed (44,45). An altered conformation of the nascent template-primer can interfere with correct nucleotide incorporation by affecting the geometry of the active site, which interferes with error discrimination by enzymes that scan for correct geometry of the minor groove (45)(46)(47)(48).…”

Section: Discussionmentioning

confidence: 99%

“…Based on structural and biochemical studies, a common fidelity mechanism involving hydrogen-bonding interactions between the polymerase and the minor groove of the template-primer has been proposed (44,45). An altered conformation of the nascent template-primer can interfere with correct nucleotide incorporation by affecting the geometry of the active site, which interferes with error discrimination by enzymes that scan for correct geometry of the minor groove (45)(46)(47)(48). It has been shown also that the minor groove of the A-form conformation of nascent primertemplate is wider, allowing easy access to the protein side chains and facilitating the interactions of the polymerase and template-primer at the polymerase active site (24,49).…”

Section: Discussionmentioning

confidence: 99%

Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine–thymine tracts

Zhang

Svarovskaia²,

Barr³

et al. 2002

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

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Using in vivo fidelity assays in which bacterial ␤-galactosidase or green fluorescent protein genes served as reporters of mutations, we have identified a murine leukemia virus (MLV) RNase H mutant (Y586F) that exhibited an increase in the retroviral mutation rate Ϸ5-fold in a single replication cycle. DNA-sequencing analysis indicated that the Y586F mutation increased the frequency of substitution mutations 17-fold within 18 nt of adenine-thymine tracts (AAAA, TTTT, or AATT), which are known to induce DNA bending. Sequence alignments indicate that MLV Y586 is equivalent to HIV-1 Y501, a component of the recently described RNase H primer grip domain, which contacts and positions the DNA primer strand near the RNase H active site. The results suggest that wild-type reverse transcriptase (RT) facilitates a specific conformation of the template-primer duplex at the polymerase active site that is important for accuracy of DNA synthesis; when an adeninethymine tract is within 18 nt of the polymerase active site, the Y586F mutant RT cannot facilitate this specific template-primer conformation, leading to an increase in the frequency of substitution mutations. These findings indicate that the RNase H primer grip can affect the template-primer conformation at the polymerase active site and that the MLV Y586 residue and template-primer conformation are important determinants of RT fidelity. R everse transcriptase (RT), which copies retroviral genomic RNA into double-stranded DNA during reverse transcription, contains two major enzymatic activities: a DNA polymerase activity that uses either RNA or DNA as a template and an RNase H activity that can degrade RNA in an RNA⅐DNA hybrid (1). The crystal structure of HIV-1 RT in complex with a template-primer and a dNTP substrate has shown that the polymerase active site and the RNase H cleavage site are separated by 18 nt of the template-primer (2). In the case of murine leukemia virus (MLV) RT, the DNA polymerase and RNase H activities reside in physically separable domains of a single monomeric protein (3).The RNase H activity of all retroviral RTs performs several functions that are essential for the viral life cycle (4), which include degradation of the RNA template after synthesis of the minusstrand DNA, generation of a specific polypurine primer from which plus-strand DNA synthesis initiates, subsequent removal of the polypurine primer to complete synthesis of the viral doublestranded DNA, and removal of the minus-strand tRNA primer (5). During minus-strand DNA synthesis, RT positions the RNase H active site and cleaves the RNA Ϸ18 nt 3Ј of the site of DNA synthesis (6).All retroviral RTs carry out error-prone DNA synthesis, in part because they lack a proofreading activity and possess a low affinity for the template (7). The low fidelity of retroviral replication is a major force for generation of high levels of variation in retroviral populations (8), which allows them to adapt quickly to changes in their environment. One important outcome of this high adaptability is t...

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“…1D). Alanine substitution for this residue dramatically decreases catalytic efficiency and fidelity (16,45,46). Thus, the modest loss of catalytic efficiency (Table 2 and 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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Base Substitution Specificity of DNA Polymerase β Depends on Interactions in the DNA Minor Groove

Cited by 66 publications

References 31 publications

Discrimination against purine–pyrimidine mispairs in the polymerase active site of DNA polymerase I: A structural explanation

Discrimination against purine–pyrimidine mispairs in the polymerase active site of DNA polymerase I: A structural explanation

Y586F mutation in murine leukemia virus reverse transcriptase decreases fidelity of DNA synthesis in regions associated with adenine–thymine tracts

DNA Polymerase β Ribonucleotide Discrimination

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