Structural Mechanism of Ribonucleotide Discrimination by a Y-Family DNA Polymerase

DNA polymerases can misinsert ribonucleotides that lead to genomic instability. DNA polymerase ␤ discourages ribonucleotide insertion with the backbone carbonyl of Tyr-271; alanine substitution of Tyr-271, but not Phe-272, resulted in a >10-fold loss in discrimination. The Y271A mutant also inserted ribonucleotides more efficiently than wild type on a variety of ribonucleoside (rNMP)-containing DNA substrates. Substituting Mn 2؉ for Mg 2؉ decreased sugar discrimination for both wildtype and mutant enzymes primarily by increasing the affinity for rCTP. This facilitated crystallization of ternary substrate complexes of both the wild-type and Y271A mutant enzymes. Crystallographic structures of Y271A-and wild type-substrate complexes indicated that rCTP is well accommodated in the active site but that O2 of rCTP and the carbonyl oxygen of Tyr-271 or Ala-271 are unusually close (ϳ2.5 and 2.6 Å , respectively). Structure-based modeling indicates that the local energetic cost of positioning these closely spaced oxygens is ϳ2.2 kcal/mol for the wild-type enzyme. Because the side chain of Tyr-271 also hydrogen bonds with the primer terminus, loss of this interaction affects its catalytic positioning. Our results support a model where DNA polymerase ␤ utilizes two strategies, steric and geometric, with a single protein residue to deter ribonucleotide insertion.

Section: Discussionmentioning

confidence: 78%

Molecular Insights into DNA Polymerase Deterrents for Ribonucleotide Insertion

Cavanaugh

Beard

Batra

et al. 2011

“…Molecular modeling suggested that this residue could interfere with binding of the rNTP 2Ј-OH and that reduction of its steric bulk accounts for the lowering of specificity in these mutants (14,28). Structural studies of a high (23) and low fidelity (29) DNA polymerase are consistent with this "steric gate" hypothesis.…”

mentioning

confidence: 79%

Structural Factors That Determine Selectivity of a High Fidelity DNA Polymerase for Deoxy-, Dideoxy-, and Ribonucleotides

Wang

Wu²,

Hellinga

et al. 2012

Background: Nucleotide selection based on sugar moiety by DNA polymerases is essential for maintaining genomic integrity. Results: Multiple polymerase conformations trap incorrect nucleotides. Conclusion: In addition to simple steric blocks, non-cognate nucleotides are prevented from incorporating by ensembles of binding sites that stabilize substrate misalignment. Significance: A structural basis has been revealed for ensembles of incorporation pathways previously defined by enzyme kinetics.

“…For example, the Y39A mutation in hpol causes the enzyme to almost totally lose its ability to discriminate between the ribose and deoxyribose sugars (17). Also, the Sulfolobus solfataricus DNA polymerase Dpo4 Y12A mutant is capable of incorporating ribonucleotides into primers, and alanine, in place of the steric gate residue Tyr-12, allows space for the 2Ј-OH of the incoming ribonucleotide, as seen in the x-ray crystal structure (16). A recently published study of yeast pol shows that mutation of the steric gate residue Phe-35 to an alanine leads to increased ability of ribonucleotide incorporation (47).…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η

Egli

Guengerich

2016

Ribonucleotides and 2-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2-OH group. Y-family human DNA polymerase (hpol ) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 10 3 -fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2-deoxyguanosine with a significant propeller twist. As a result, the 2-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and P␣ of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.RNA and DNA are fundamental to life in all forms. The two nucleic acid polymers are composed of ribonucleotides and 2Ј-deoxyribonucleotides as the basic units, respectively. However, ribonucleotides have been found in DNA; they constitute a large proportion of the "DNA lesions" in the genome. In mice, they have been shown to be collectively the most frequently occurring DNA lesions, even more than abasic sites and 7,8-dihydro-8-oxo-2Ј-deoxyguanosine (8-oxodG).2 The presence of ribonucleotides in DNA increases the possibility of spontaneous hydrolysis, causing DNA to break more frequently. These ribonucleotides are mainly removed from DNA by the RNase H2 pathway (1-5). DNA polymerases introduce ribonucleotides into DNA by misinserting them (6). Concentrations of cellular rNTPs are 1-6 orders of magnitude higher than those of dNTPs, depending on the cell type and the stage of the cell cycle (6 -9). To discriminate dNTP from rNTP, a steric gate residue (typically a tyrosine or phenylalanine) is generally conserved in DNA polymerases and thought to create a clash with the extra hydroxyl group of the incoming rNTP (10 -17). Although limited in extent, ribonucleotide incorporation has still been observed by a variety of DNA polymerases, from replicative ones with relatively high fidelity and small active sites (e.g. pol ⑀ and pol ␦) to error-prone X-family pol and pol ␤ and Y-family pol (6,8,(17)(18)(19)(20)(21).Compared with other DNA polymerases, those in the Yfamily are known for high ...