Structure–function analysis of ribonucleotide bypass by B family DNA replicases

Clausen, Anders R.; Murray, Michael S.; Passer, Andrew R.; Pedersen, Lars C.; Kunkel, Thomas A.

doi:10.1073/pnas.1309119110

Cited by 45 publications

(59 citation statements)

References 49 publications

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“…Because rNTPs (including 8-oxorGTP) can be incorporated into DNA during replication, ribonucleotides, if not repaired, are problematic DNA lesions due to its potential to retard DNA replication (1,28). However, the ability of pols ␣, , and to bypass the ribonucleotides is still unclear.…”

Section: Discussionmentioning

confidence: 99%

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Impact of Ribonucleotide Backbone on Translesion Synthesis and Repair of 7,8-Dihydro-8-oxoguanine

Sassa

Çağlayan

Rodriguez

et al. 2016

Journal of Biological Chemistry

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Edited by Patrick SungNumerous ribonucleotides are incorporated into the genome during DNA replication. Oxidized ribonucleotides can also be erroneously incorporated into DNA. Embedded ribonucleotides destabilize the structure of DNA and retard DNA synthesis by DNA polymerases (pols), leading to genomic instability. Mammalian cells possess translesion DNA synthesis (TLS) pols that bypass DNA damage. The mechanism of TLS and repair of oxidized ribonucleotides remains to be elucidated. To address this, we analyzed the miscoding properties of the ribonucleotides riboguanosine (rG) and 7,8-dihydro-8-oxo-riboguanosine (8-oxo-rG) during TLS catalyzed by the human TLS pols and in vitro. The primer extension reaction catalyzed by human replicative pol ␣ was strongly blocked by 8-oxo-rG. pol inefficiently bypassed rG and 8-oxo-rG compared with dG and 7,8-dihydro-8-oxo-2-deoxyguanosine (8-oxo-dG), whereas pol easily bypassed the ribonucleotides. pol ␣ exclusively inserted dAMP opposite 8-oxo-rG. Interestingly, pol preferentially inserted dCMP opposite 8-oxo-rG, whereas the insertion of dAMP was favored opposite 8-oxo-dG. In addition, pol accurately bypassed 8-oxo-rG. Furthermore, we examined the activity of the base excision repair (BER) enzymes 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease 1 on the substrates, including rG and 8-oxorG. Both BER enzymes were completely inactive against 8-oxo-rG in DNA. However, OGG1 suppressed 8-oxo-rG excision by RNase H2, which is involved in the removal of ribonucleotides from DNA. These results suggest that the different sugar backbones between 8-oxo-rG and 8-oxo-dG alter the capacity of TLS and repair of 8-oxoguanine.

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Section: Discussionmentioning

confidence: 99%

“…As with various DNA damages, ribonucleotides in DNA templates retard DNA synthesis in vitro by replicative pols ␦ and ⑀ but not by TLS pol (1,28,29). Thus, TLS pols are important for the damage tolerance of embedded ribonucleotides.…”

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confidence: 99%

Impact of Ribonucleotide Backbone on Translesion Synthesis and Repair of 7,8-Dihydro-8-oxoguanine

Sassa

Çağlayan

Rodriguez

et al. 2016

Journal of Biological Chemistry

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“…Similarly, in DNA polymerase Dpo4, a pol homolog from S. solfataricus, the steric gate residue Tyr-12 is accompanied by Tyr-81 that serves as the second line of defense (50). Conversely, the replicative B-family bacteriophage RB69 uses a single DNA polymerase steric gate residue, Tyr-416, packed against an ␣-helix (19).…”

Section: ј-Oh Of the Incoming Ribonucleotide Leads To A Propeller Twmentioning

confidence: 99%

“…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 misinsertion rates and tolerance of DNA adducts in the template strand by relying on their spacious active sites (22-24). Y-family human DNA polymerase (hpol ) is of particular interest, as it is the only known DNA polymerase directly related to a human genetic disorder, mainly due to its unique role in translesion synthesis past the UVinduced DNA lesion cyclobutane pyrimidine dimer (CPD).…”

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Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η

Egli

Guengerich

2016

Journal of Biological Chemistry

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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 ...

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Structural Impact of Single Ribonucleotide Residues in DNA

et al. 2016

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Single ribonucleotide intrusions represent the most common nonstandard nucleotide type found incorporated in genomic DNA, yet little is known of their structural impact. This lesion incurs genomic instability in addition to affecting the physical properties of the DNA. To probe for structural and dynamic effects of single ribonucleotides in various sequence contexts-AxC, CxG, and GxC, where x=rG or dG-we report the structures of three single-ribonucleotide-containing DNA duplexes and the corresponding DNA controls. The lesion subtly and locally perturbs the structure asymmetrically on the 3' side of the lesion in both the riboguanosine-containing and the complementary strand of the duplex. The perturbations are mainly restricted to the sugar and phosphodiester backbone. The ribose and 3'-downstream deoxyribose units are predominately in N-type conformation; backbone torsion angles ϵ and/or ζ of the ribonucleotide or upstream deoxyribonucleotide are affected. Depending on the flanking sequences, the C2'-OH group forms hydrogen bonds with the backbone, 3'-neighboring base, and/or sugar. Interestingly, even in similar purine-rG-pyrimidine environments (A-rG-C and G-rG-C), a riboguanosine unit affects DNA in a distinct manner and manifests different hydrogen bonds, which makes generalizations difficult.

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Structure–function analysis of ribonucleotide bypass by B family DNA replicases

Cited by 45 publications

References 49 publications

Impact of Ribonucleotide Backbone on Translesion Synthesis and Repair of 7,8-Dihydro-8-oxoguanine

Impact of Ribonucleotide Backbone on Translesion Synthesis and Repair of 7,8-Dihydro-8-oxoguanine

Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η

Structural Impact of Single Ribonucleotide Residues in DNA

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