Networked Communication between Polymerase and Exonuclease Active Sites in Human Mitochondrial DNA Polymerase

Sowers, Mark L.; Anderson, Andrew P; Wrabl, James O.; Yin, Yajiang

doi:10.1021/jacs.9b04655

Cited by 5 publications

(9 citation statements)

References 59 publications

(108 reference statements)

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“…Quantification of primer degradation for the POLIB variants demonstrated that catalytic mutations in IBPol-impacted the rate of DNA degradation about 2-fold, suggesting that there may be crosstalk between the Pol and Exo domains of POLIB, a characteristic described in other well-characterized DNA polymerases such as human Pol g. 28 No 5’-3’ exonuclease activity was detected when using 3’ fluorescein (6-FAM)-labeled single stranded or double stranded DNA substrates compared to the T5 exo control (Figure S7).…”

Section: Resultsmentioning

confidence: 91%

“…This is a phenomenon described in other pols such as human Pol g and may be a more dramatic effect in POLIB due to the divergent arrangement of the two functional domains. 28 In recombinant IBWT, exo activity unexpectedly prevails over pol activity at higher pH conditions (Figure 5). These more basic conditions are likely closer to the physiological pH of the mitochondrial matrix, based on what has been reported in other systems.…”

Section: Discussionmentioning

confidence: 93%

“…11,12,26 Additionally, proofreading Family A members have the exo domain located under the palm subdomain ( E. coli Pol I, Pol γ). 27,28 Thus far, POLIB is divergent from all other Family A proteins containing a unique substitution within Motif C, and the largest thumb insertion with similarity to an exo domain. Interestingly, these divergent features are conserved among all the POLIB sequences from Kinetoplastid organisms sequenced to date (Figure S5).…”

Section: Resultsmentioning

confidence: 99%

“…This is a phenomenon described in other pols such as human Pol g and may be a more dramatic effect in POLIB due to the divergent arrangement of the two functional domains. 28…”

Section: Discussionmentioning

confidence: 99%

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Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Thumb Insertion That Confers Dominant Exonuclease Activity

Klingbeil

Nelson²,

Frost

et al. 2022

Preprint

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Trypanosoma brucei and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent and essential for parasite survival. POLIB is one of three Family A DNA polymerases independently essential to maintain the kDNA network. However, the division of labor among the paralogs, particularly which might fulfill the role of a replicative, proofreading enzyme remains enigmatic. De novo modeling of POLIB revealed a structure divergent from all other Family A polymerases in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3'-5' exonucleases. Here we demonstrate recombinant POLIB 3'-5' exonuclease prefers DNA vs. RNA substrates and degrades single- and double-stranded DNA in a non processive manner. The exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB is able to extend an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either primer. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborate the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The unique POLIB thumb insertion influences the balance between polymerase and exonuclease activity and provides another example of exquisite diversity among DNA polymerases for specialized function.

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Section: Resultsmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 93%

Section: Resultsmentioning

confidence: 99%

“…This is a phenomenon described in other pols such as human Pol g and may be a more dramatic effect in POLIB due to the divergent arrangement of the two functional domains. 28…”

Section: Discussionmentioning

confidence: 99%

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Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Thumb Insertion That Confers Dominant Exonuclease Activity

Klingbeil

Nelson²,

Frost

et al. 2022

Preprint

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show abstract

“…3A). Mutations in the polymerization domain of human and yeast mitochondrial DNAPs can alter the exonuclease active site [32,33], raising the possibility that loss of the -hairpin indirectly affects the shuttling of Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200922/915490/bcj-2020-0922.pdf by guest on 28 June 2021 the primer strand into the exonuclease domain of T7 DNAP. The decreased polymerase activity observed by the T7 DNAP-Δ5 -hairpin is corroborated in a strand-displacement assay using a gapped substrate that allows DNA synthesis to commence prior to encountering a blocking oligonucleotide of 25 nts that requires strand displacement for further synthesis.…”

Section: An Extended -Hairpin In the Finger Subdomain Guides The Template Into The Active Site And Promotes Strand-displacementmentioning

confidence: 99%

Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site

Juarez-Quintero¹,

Peralta-Castro²,

Cardoza

et al. 2021

Biochemical Journal

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The crystal structure of full-length T7 DNA polymerase in complex with its processivity factor thioredoxin and double-stranded DNA in the polymerization active site exhibits two novel structural motifs in family-A DNA polymerases: an extended b-hairpin at the fingers subdomain, that interacts with the DNA template strand downstream the primer-terminus, and a helix-loop-helix motif (insertion1) located between residues 102 to 122 in the exonuclease domain. The extended b-hairpin is involved in nucleotide incorporation on substrates with 5'-overhangs longer than 2 nucleotides, suggesting a role in stabilizing the template strand into the polymerization domain. Our biochemical data reveal that insertion1 of the exonuclease domain makes stabilizing interactions that facilitate proofreading by shuttling the primer strand into the exonuclease active site. Overall, our studies evidence conservation of the 3'-5' exonuclease domain fold between family-A DNA polymerases and highlight the modular architecture of T7 DNA polymerase. Our data suggest that the intercalating b-hairpin guides the template-strand into the polymerization active site after the T7 primase-helicase unwinds the DNA double helix ameliorating the formation of secondary structures and decreasing the appearance of indels

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Remdesivir triphosphate blocks DNA synthesis and increases exonucleolysis by the replicative mitochondrial DNA polymerase, Pol γ

et al. 2021

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Networked Communication between Polymerase and Exonuclease Active Sites in Human Mitochondrial DNA Polymerase

Cited by 5 publications

References 59 publications

Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Thumb Insertion That Confers Dominant Exonuclease Activity

Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Thumb Insertion That Confers Dominant Exonuclease Activity

Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site

Remdesivir triphosphate blocks DNA synthesis and increases exonucleolysis by the replicative mitochondrial DNA polymerase, Pol γ

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