DNA polymerase 3′→5′ exonuclease activity: Different roles of the beta hairpin structure in family-B DNA polymerases

Darmawan, Hariyanto; Harrison, Melissa; Reha-Krantz, Linda J.

doi:10.1016/j.dnarep.2015.02.014

Cited by 16 publications

(12 citation statements)

References 65 publications

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“…A glycine in this hairpin has been substituted with a serine, which led to a mutant polymerase with an increased tolerance for replication mistakes, because the strand separation cannot be stabilized in a way to ensure sufficient exonuclease activity (54). However, whether this hairpin plays a similar role in other family B DNA polymerases remains speculative, as hairpins among these polymerases show variations in their amino acid sequence (56). Moreover, there is evidence that the hairpin is not generally required for proofreading by family B DNA polymerases, as shown for Saccharomyces cerevisiae polymerases δ (57) and ϵ (58).…”

Section: Discussionmentioning

confidence: 99%

5-methylcytosine-sensitive variants ofThermococcus kodakaraensisDNA polymerase

Huber¹,

Watzdorf²,

Marx³

2016

Nucleic Acids Res

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DNA methylation of cytosine in eukaryotic cells is a common epigenetic modification, which plays an important role in gene expression and thus affects various cellular processes like development and carcinogenesis. The occurrence of 5-methyl-2′-deoxycytosine (5mC) as well as the distribution pattern of this epigenetic marker were shown to be crucial for gene regulation and can serve as important biomarkers for diagnostics. DNA polymerases distinguish little, if any, between incorporation opposite C and 5mC, which is not surprising since the site of methylation is not involved in Watson–Crick recognition. Here, we describe the development of a DNA polymerase variant that incorporates the canonical 2′-deoxyguanosine 5′-monophosphate (dGMP) opposite C with higher efficiency compared to 5mC. The variant of Thermococcus kodakaraensis (KOD) exo- DNA polymerase was discovered by screening mutant libraries that were built by rational design. We discovered that an amino acid substitution at a single site that does not directly interact with the templating nucleobase, may alter the ability of the DNA polymerase in processing C in comparison to 5mC. Employing these findings in combination with a nucleotide, which is fluorescently labeled at the terminal phosphate, indicates the potential use of the mutant DNA polymerase in the detection of 5mC.

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Section: Discussionmentioning

confidence: 99%

5-methylcytosine-sensitive variants ofThermococcus kodakaraensisDNA polymerase

Huber¹,

Watzdorf²,

Marx³

2016

Nucleic Acids Res

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“…Although the β-hairpin loop is located in the same position within their exonuclease domain, not always play the same role as in RB69 gp43 and T4 gp43 (Table 1 ). DNA pol δ has similarly placed β-hairpin loop, but as it was demonstrated very recently, DNA pol δ does not need the hairpin for proofreading, but β-hairpin loop is required for optimum DNA replication efficiency, because its role is to stabilize polymerase complexes (Darmawan et al 2015 ). The β-hairpin loop is truncated in pol ε, is too short to contact the DNA, and presumably is not involved in active site switching (Ganai et al 2015 ).…”

Section: Replication Fidelitymentioning

confidence: 99%

Fidelity of DNA replication—a matter of proofreading

2018

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DNA that is transmitted to daughter cells must be accurately duplicated to maintain genetic integrity and to promote genetic continuity. A major function of replicative DNA polymerases is to replicate DNA with the very high accuracy. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and postreplicative DNA mismatch repair (MMR). Proofreading activity that assists most of the replicative polymerases is responsible for removal of incorrectly incorporated nucleotides from the primer terminus before further primer extension. It is estimated that proofreading improves the fidelity by a 2–3 orders of magnitude. The primer with the incorrect terminal nucleotide has to be moved to exonuclease active site, and after removal of the wrong nucleotide must be transferred back to polymerase active site. The mechanism that allows the transfer of the primer between pol and exo site is not well understood. While defects in MMR are well known to be linked with increased cancer incidence only recently, the replicative polymerases that have alterations in the exonuclease domain have been associated with some sporadic and hereditary human cancers. In this review, we would like to emphasize the importance of proofreading (3′-5′ exonuclease activity) in the fidelity of DNA replication and to highlight what is known about switching from polymerase to exonuclease active site.

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“…The thumb domain interacts with the primer–template complex. The architecture of the type B DNApol harbors a 3′-5′ exonuclease domain whose role is to correct misincorporated nucleotides and to maintain the fidelity and integrity of the newly formed DNA molecules (30,31). Interestingly, the HSV-1 DNApol has an extra domain, the pre-NH 2 -terminal domain, according to the three dimensional structure published by Liu et al (32).…”

Section: Structural Features Of Dna Polymerase B Familymentioning

confidence: 99%

“…The bacteriophage RB69 DNApol is one of the most studied at the structural and functional levels, and there are currently 122 entries in the protein data bank (http://www.rcsb.org/pdb/results/results.do?outformat=&qrid=C9789076&tabtoshow=Current) (30,36–40). Although RB69 DNApol lacks the pre-NH 2 -terminal domain, it is a good surrogate model for herpesvirus DNApol, especially regarding structural changes involved in catalysis and ligand binding (DNA, dNTPs) (36).…”

Section: Structural Features Of Dna Polymerase B Familymentioning

confidence: 99%

Distribution and effects of amino acid changes in drug-resistant α and β herpesviruses DNA polymerase

Topalis¹,

Gillemot²,

Snoeck³

et al. 2016

Nucleic Acids Res

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Emergence of drug-resistance to all FDA-approved antiherpesvirus agents is an increasing concern in immunocompromised patients. Herpesvirus DNA polymerase (DNApol) is currently the target of nucleos(t)ide analogue-based therapy. Mutations in DNApol that confer resistance arose in immunocompromised patients infected with herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV), and to lesser extent in herpes simplex virus 2 (HSV-2), varicella zoster virus (VZV) and human herpesvirus 6 (HHV-6). In this review, we present distinct drug-resistant mutational profiles of herpesvirus DNApol. The impact of specific DNApol amino acid changes on drug-resistance is discussed. The pattern of genetic variability related to drug-resistance differs among the herpesviruses. Two mutational profiles appeared: one favoring amino acid changes in the Palm and Finger domains of DNApol (in α-herpesviruses HSV-1, HSV-2 and VZV), and another with mutations preferentially in the 3′-5′ exonuclease domain (in β-herpesvirus HCMV and HHV-6). The mutational profile was also related to the class of compound to which drug-resistance emerged.

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DNA polymerase 3′→5′ exonuclease activity: Different roles of the beta hairpin structure in family-B DNA polymerases

Cited by 16 publications

References 65 publications

5-methylcytosine-sensitive variants ofThermococcus kodakaraensisDNA polymerase

5-methylcytosine-sensitive variants ofThermococcus kodakaraensisDNA polymerase

Fidelity of DNA replication—a matter of proofreading

Distribution and effects of amino acid changes in drug-resistant α and β herpesviruses DNA polymerase

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