Excessive excision of correct nucleotides during
            <scp>DNA</scp>
            synthesis explained by replication hurdles

Singh, Anupam; Pandey, Manjula; Nandakumar, Divya; Raney, Kevin D.; Yin, Yajiang; Patel, Smita S.

doi:10.15252/embj.2019103367

Cited by 16 publications

(14 citation statements)

References 78 publications

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“…their active site) which establishes the Z-vs-A preference, but rather the ability to promote the transition from the extending mode to the proofreading mode in the kinetic scheme of the polymerase reaction (Figure 6A ). This ability has recently been shown to occur much more frequently than previously thought, including for correctly incorporated nucleotides ( 60 ). It relies in part on polymerase backtracking, which occurs just before the 3′-end of the newly synthetized strand is sent to the exonuclease active site; it is well described for DNA-dependent RNA polymerases ( 61 ), and structural evidence for this step has been found recently on DNA-dependent DNA polymerases ( 62 ).…”

Section: Discussionmentioning

confidence: 99%

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8

Czernecki

Romoli

et al. 2021

Nucleic Acids Research

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All genetic information in cellular life is stored in DNA copolymers composed of four basic building blocks (ATGC-DNA). In contrast, a group of bacteriophages belonging to families Siphoviridae and Podoviridae has abandoned the usage of one of them, adenine (A), replacing it with 2-aminoadenine (Z). The resulting ZTGC-DNA is more stable than its ATGC-DNA counterpart, owing to the additional hydrogen bond present in the 2-aminoadenine:thymine (Z:T) base pair, while the additional amino group also confers resistance to the host endonucleases. Recently, two classes of replicative proteins found in ZTGC-DNA-containing phages were characterized and one of them, DpoZ from DNA polymerase A (PolA) family, was shown to possess significant Z-vs-A specificity. Here, we present the crystallographic structure of the apo form of DpoZ of vibriophage ϕVC8, composed of the 3′-5′ exonuclease and polymerase domains. We captured the enzyme in two conformations that involve the tip of the thumb subdomain and the exonuclease domain. We highlight insertions and mutations characteristic of ϕVC8 DpoZ and its close homologues. Through mutagenesis and functional assays we suggest that the preference of ϕVC8 DpoZ towards Z relies on a polymerase backtracking process, more efficient when the nascent base pair is A:T than when it is Z:T.

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Section: Discussionmentioning

confidence: 99%

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8

Czernecki

Romoli

et al. 2021

Nucleic Acids Research

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“…This is in agreement with the increased numbers of ρ ° cells seen at 37°C (Baruffini et al, 2007), in which case excess DNA degradation could contribute to the loss of mtDNA. In addition, as shown with T7 DNA polymerase, exonuclease activity can be increased in the presence of “replication hurdles,” such as DNA secondary structure or weakened interactions with helicases or other components of the replication machinery (Singh et al, 2020). These structural issues could impact the truncation variants of Mip1 more severely, leading to increased DNA degradation.…”

Section: Discussionmentioning

confidence: 99%

A non‐radioactive DNA synthesis assay demonstrates that elements of the Sigma 1278b Mip1 mitochondrial DNA polymerase domain and C‐terminal extension facilitate robust enzyme activity

Young

Imperial

Lakhi

et al. 2021

Yeast

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The yeast DNA polymerase gamma, Mip1, is a useful tool to investigate the impact of orthologous human disease variants on mitochondrial DNA (mtDNA) replication. However, Mip1 is characterized by a C‐terminal extension (CTE) that is not found on orthologous metazoan DNA polymerases, and the CTE is required for robust enzymatic activity. Two MIP1 alleles exist in standard yeast strains, encoding Mip1[S] or Mip1[Σ]. Mip1[S] is associated with reduced mtDNA stability and increased error rates in vivo. Although the Mip1[S] allele was initially identified in S288c, the Mip1[Σ] allele is widely present among available yeast genome sequences, suggesting that it is the wild‐type (WT) allele. We developed a novel non‐radioactive polymerase gamma assay to assess Mip1 functioning at its intracellular location, the mitochondrial membrane. Membrane fractions were isolated from yeast cells expressing full‐length or CTE truncation variants of Mip1[S] or a chimeric Mip1[S] isoform harboring the Mip1[Σ]‐specific T661 residue (cMip1 T661). Relative incorporation of digoxigenin (DIG)‐11‐deoxyuridine monophosphate (DIG‐dUMP) by cMip1 T661 was higher than that by Mip1[S]. A cMip1 T661variant lacking 175 C‐terminal residues maintained WT levels of DIG‐dUMP incorporation, whereas the C‐terminal variant lacking 205 residues displayed a significant decrease in incorporation. Newly synthesized DIG‐labeled DNA decreased during later phases of reactions carried out at 37°C, suggesting temperature‐sensitive destabilization of the polymerase domain and/or increased shuttling of the nascent DNA into the exonuclease domain. Comparative analysis of Mip1 enzyme functions using our novel assay has further demonstrated the importance of the CTE and T661 encoded by MIP1[Σ] in yeast mtDNA replication.

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“…This allowed further polymerase activity and chain elongation after GCV-TP incorporation and generation of full-length gene products [66]. During DNA synthesis, also correctly incorporated nucleotides are frequently excised [84]. Thus, we can hypothesize that the mutation Y383S GCV affects the exonuclease activity, reducing excision of correct nucleotides and/or elongating the viral DNA after antiviral incorporation, consequently leading to a growth advantage of the drug-resistant viral clone over the wild-type virus.…”

Section: Discussionmentioning

confidence: 99%

An MHV-68 Mutator Phenotype Mutant Virus, Confirmed by CRISPR/Cas9-Mediated Gene Editing of the Viral DNA Polymerase Gene, Shows Reduced Viral Fitness

Trompet

Temblador

Gillemot

et al. 2021

Viruses

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Drug resistance studies on human γ-herpesviruses are hampered by the absence of an in vitro system that allows efficient lytic viral replication. Therefore, we employed murine γ-herpesvirus-68 (MHV-68) that efficiently replicates in vitro as a model to study the antiviral resistance of γ-herpesviruses. In this study, we investigated the mechanism of resistance to nucleoside (ganciclovir (GCV)), nucleotide (cidofovir (CDV), HPMP-5azaC, HPMPO-DAPy) and pyrophosphate (foscarnet (PFA)) analogues and the impact of these drug resistance mutations on viral fitness. Viral fitness was determined by dual infection competition assays, where MHV-68 drug-resistant viral clones competed with the wild-type virus in the absence and presence of antivirals. Using next-generation sequencing, the composition of the viral populations was determined at the time of infection and after 5 days of growth. Antiviral drug resistance selection resulted in clones harboring mutations in the viral DNA polymerase (DP), denoted Y383SGCV, Q827RHPMP-5azaC, G302WPFA, K442TPFA, G302W+K442TPFA, C297WHPMPO-DAPy and C981YCDV. Without antiviral pressure, viral clones Q827RHPMP-5azaC, G302WPFA, K442TPFA and G302W+K442TPFA grew equal to the wild-type virus. However, in the presence of antivirals, these mutants had a growth advantage over the wild-type virus that was moderately to very strongly correlated with antiviral resistance. The Y383SGCV mutant was more fit than the wild-type virus with and without antivirals, except in the presence of brivudin. The C297W and C981Y changes were associated with a mutator phenotype and had a severely impaired viral fitness in the absence and presence of antivirals. The mutator phenotype caused by C297W in MHV-68 DP was validated by using a CRISPR/Cas9 genome editing approach.

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Excessive excision of correct nucleotides during DNA synthesis explained by replication hurdles

Cited by 16 publications

References 78 publications

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8

Structural dynamics and determinants of 2-aminoadenine specificity in DNA polymerase DpoZ of vibriophage ϕVC8

A non‐radioactive DNA synthesis assay demonstrates that elements of the Sigma 1278b Mip1 mitochondrial DNA polymerase domain and C‐terminal extension facilitate robust enzyme activity

An MHV-68 Mutator Phenotype Mutant Virus, Confirmed by CRISPR/Cas9-Mediated Gene Editing of the Viral DNA Polymerase Gene, Shows Reduced Viral Fitness

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