Yeast DNA polymerase η makes functional contacts with the DNA minor groove only at the incoming nucleoside triphosphate

Washington, M. Todd; Wolfle, William T.; Spratt, Thomas E.; Prakash, Louise; Prakash, Satya

doi:10.1073/pnas.0837578100

Cited by 39 publications

(41 citation statements)

References 33 publications

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“…However, the geometry of the incipient base pair is also likely to be important for Pol. This is because for certain base pairs, Pol preferentially incorporates the correct nucleotide several thousandfold more efficiently than the incorrect nucleotide (45) and because Pol requires minor-groove interactions between the protein and the incoming nucleotide substrate (47). The latter observation raises the possibility that this interaction provides for a "geometry-sensing" mechanism similar to those proposed for classical DNA polymerases.…”

Section: Discussionmentioning

confidence: 96%

Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase η

Washington

Helquist

Kool

et al. 2003

Molecular and Cellular Biology

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Classical high-fidelity DNA polymerases discriminate between the correct and incorrect nucleotides by using geometric constraints imposed by the tight fit of the active site with the incipient base pair. Consequently, Watson-Crick (W-C) hydrogen bonding between the bases is not required for the efficiency and accuracy of DNA synthesis by these polymerases. DNA polymerase (Pol) is a low-fidelity enzyme able to replicate through DNA lesions. Using difluorotoluene, a nonpolar isosteric analog of thymine unable to form W-C hydrogen bonds with adenine, we found that the efficiency and accuracy of nucleotide incorporation by Pol are severely impaired. From these observations, we suggest that W-C hydrogen bonding is required for DNA synthesis by Pol; in this regard, Pol differs strikingly from classical high-fidelity DNA polymerases.Classical DNA polymerases, such as T7 and Escherichia coli Klenow, synthesize DNA with high fidelity, and they are unable to replicate through DNA lesions (7). By contrast, the eukaryotic polymerase (Pol) is a low-fidelity enzyme (16,28,45) with the ability to replicate through DNA lesions. Pol is unique among eukaryotic DNA polymerases in its proficient ability to replicate through UV-induced cyclobutane pyrimidine dimers (CPDs) (15). Remarkably, both yeast and human Pol insert A's opposite the two T's of a cis-syn thyminethymine (TT) dimer with the same efficiency and accuracy as they insert A's opposite undamaged template bases (16,44). In addition to TT dimers, UV light also induces the formation of CPDs at 5Ј-TC-3Ј and 5Ј-CC-3Ј sites, and genetic studies with Saccharomyces cerevisiae have indicated a role of Pol in the accurate bypass of these lesions as well (48). Because of its role in promoting the error-free bypass of CPDs, inactivation of Pol in humans causes UV hypermutability (43) and results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum (14,27). Yeast Pol also replicates through an 8-oxoguanine (8-oxoG) lesion efficiently and accurately, and genetic studies with S. cerevisiae have corroborated the requirement of Pol in the error-free bypass of this DNA lesion (11).From studies done with nonpolar isosteric analogs of natural DNA bases which lack the ability to form Watson-Crick (W-C) hydrogen (H) bonds, it has been concluded that W-C H bonding is not needed for DNA synthesis by classical high-fidelity DNA polymerases (30,32,33); rather, the geometric fit of the incoming nucleotide with the templating base within the polymerase active site is the principal determinant of the efficiency and fidelity of DNA synthesis in these polymerases (7,9,22). Here we examine the effects of difluorotoluene (F), which is virtually identical in shape, size, and conformation to thymine (T) but lacks the ability to form W-C H bonds with adenine (A) (Fig. 1A), on DNA synthesis by yeast Pol. Because of the inability of F to form W-C H bonds in water (37, 38), this compound is ideal for examining the relative importance of hydrogen bonds and steric effects on DNA synthesis. P...

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Section: Discussionmentioning

confidence: 96%

Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase η

Washington

Helquist

Kool

et al. 2003

Molecular and Cellular Biology

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“…Synthetic oliogodeoxynucleotide template and primers were used to prepare the nondamaged DNA substrates. The 3-deazaguanine (3DG)-and F-containing DNA substrates were synthesized and purified as described previously (37,45). For the 3DG-containing substrates, the following template/primer pairs were used.…”

Section: Methodsmentioning

confidence: 99%

Evidence for a Watson-Crick Hydrogen Bonding Requirement in DNA Synthesis by Human DNA Polymerase κ

Wolfle

Washington

Kool

et al. 2005

Molecular and Cellular Biology

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The efficiency and fidelity of nucleotide incorporation by high-fidelity replicative DNA polymerases (Pols) are governed by the geometric constraints imposed upon the nascent base pair by the active site. Consequently, these polymerases can efficiently and accurately replicate through the template bases which are isosteric to natural DNA bases but which lack the ability to engage in Watson-Crick (W-C) hydrogen bonding. DNA synthesis by Pol, a low-fidelity polymerase able to replicate through DNA lesions, however, is inhibited in the presence of such an analog, suggesting a dependence of this polymerase upon W-C hydrogen bonding. Here we examine whether human Pol, which differs from Pol in having a higher fidelity and which, unlike Pol, is inhibited at inserting nucleotides opposite DNA lesions, shows less of a dependence upon W-C hydrogen bonding than does Pol. We find that an isosteric thymidine analog is replicated with low efficiency by Pol, whereas a nucleobase analog lacking minor-groove H bonding potential is replicated with high efficiency. These observations suggest that both Pol and Pol rely on W-C hydrogen bonding for localizing the nascent base pair in the active site for the polymerization reaction to occur, thus overcoming these enzymes' low geometric selectivity.Classical DNA polymerases (Pols) replicate DNA with a high fidelity and are unable to replicate through DNA-distorting lesions. From studies with nonpolar, isosteric analogs, which lack the ability to form canonical Watson-Crick (W-C) hydrogen bonds, it has been concluded that W-C H bonding is not the determining factor for the efficiency and accuracy of nucleotide incorporation in high-fidelity polymerases such as T7 and Escherichia coli Pol I (20, 23, 24); rather, it is apparently the geometric fit within the active site of the incoming nucleoside triphosphate with the templating nucleotide that governs polymerase efficiency and accuracy (3,5,13,15).Members of the Y family of DNA polymerases replicate DNA with a low fidelity, and unlike the classical polymerases, they are able to replicate through DNA lesions (28, 29). Humans have four Y family Pols: , , , and Rev1. Of these, Rev1 is a highly specialized polymerase which predominantly incorporates a C opposite template G and also opposite an abasic site (8,26), and genetic studies of Saccharomyces cerevisiae have suggested that a major role of Rev1 is to act as an assembly factor in Pol-dependent lesion bypass (28). Although not as extreme in its nucleotide insertion specificity as Rev1, Pol incorporates nucleotides opposite the four different template bases with very different efficiencies and fidelities, and it incorporates nucleotides opposite template purines with a much higher efficiency and fidelity than opposite template pyrimidines (6,11,34,39,46). From the ternary crystal structure of Pol, with a templating A and an incoming dTTP, it has been determined that unlike all other DNA polymerases, which impose Watson-Crick base pairing in their active site, Pol uses Hoogsteen base pair...

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“…The concentrations of oligodeoxynucleotides were determined from the absorbance at 260 nm, using the method of Borer (30) in which it was assumed that the spectroscopic properties of 3DG were identical to G. The primer was 32 …”

Section: General-[mentioning

confidence: 99%

“…Arg 668 has been shown to be important to catalysis and fidelity of DNA replication (16,20,28,31), but the overall structure of the protein appears unaffected by the substitution (28). 3DG has been used to examine minor groove interactions and is a good substrate at some sites in the DNA but not others and therefore, is a good mimic of guanine (24,32). Several nucleotide analogs in which the deoxyribose moiety was replaced with a carbocycle have been examined and they inhibit DNA synthesis by competitive inhibition or chain termination depending on the polymerase (33)(34)(35).…”

Section: P-labeled With [␥-mentioning

confidence: 99%

Escherichia coli DNA Polymerase I (Klenow Fragment) Uses a Hydrogen-bonding Fork from Arg668 to the Primer Terminus and Incoming Deoxynucleotide Triphosphate to Catalyze DNA Replication

Meyer

Blandino

Spratt

2004

Journal of Biological Chemistry

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Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication. In particular, Arg 668 of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus. We investigated the interaction between Arg 668 and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP. Hydrogen bonds from Arg 668 were probed with the site-specific mutant R668A. Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus. Hydrogen bonds from the incoming dNTP were probed with (1R,3R,4R)-1-[3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl]uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group. We found that the pre-steady-state parameter k pol was decreased 1,600 to 2,000-fold with each of the single substitutions. When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in k pol . These results are consistent with a hydrogenbonding fork from Arg 668 to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication.The high fidelity of DNA replication synthesis is accomplished despite the similarity in energy between correctly and incorrectly paired bases (1, 2). Since inter-strand hydrogen bonds cannot account for this selectivity (3, 4), other mechanisms have been proposed to supply the fidelity (reviewed in Ref. 5). These include solvation (6), base stacking (7, 8), steric exclusion (3, 4, 9), and minor groove binding (10). These mechanisms are not mutually exclusive and may all enhance selection for the correct base pairs.The minor groove has been suggested to be a site by which polymerases can check geometry because the O 2 -position of pyrimidines and the N-3-position of purines occupy similar spatial orientations as well as being hydrogen bond acceptors (10). Crystal structures of DNA polymerases bound to DNA have shown that there are many interactions between the protein and the minor groove of DNA (11-15). Site-directed mutagenesis studies have implicated several amino acid residues as being important for catalysis and fidelity of DNA replication (15)(16)(17)(18)(19)(20). Similarly, the use of purine analogs such as 3-deazaadenine (21, 22), 3-deazaguanine (3DG) 1 (23, 24), 9-methyl-1H-imidazo[4,5-b]pyridine and 4-methylbenzimidazole (25,26), and the pyrimidine analogs difluorotoluene (25), 2-aminopyridine, and 3-methyl-2-pyridone (27) also have implicated the minor groove of the DNA as being crucial.X-ray crystallographic studies of polymerases that have a structure similar to that of KF Ϫ (Taq (13) Bacillus stearothermophilus (12) and T7 (14)...

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Yeast DNA polymerase η makes functional contacts with the DNA minor groove only at the incoming nucleoside triphosphate

Cited by 39 publications

References 33 publications

Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase η

Requirement of Watson-Crick Hydrogen Bonding for DNA Synthesis by Yeast DNA Polymerase η

Evidence for a Watson-Crick Hydrogen Bonding Requirement in DNA Synthesis by Human DNA Polymerase κ

Escherichia coli DNA Polymerase I (Klenow Fragment) Uses a Hydrogen-bonding Fork from Arg668 to the Primer Terminus and Incoming Deoxynucleotide Triphosphate to Catalyze DNA Replication

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