Preferential Incorporation of G Opposite Template T by the Low-Fidelity Human DNA Polymerase ι

ABSTRACT DNA polymerase activity is essential for replication, recombination, repair, and mutagenesis. All DNA polymerases studied so far from any biological source synthesize DNA by the Watson-Crick base-pairing rule, incorporating A, G, C, and T opposite the templates T, C, G, and A, respectively. Non-Watson-Crick base pairs would lead to mutations. In this report, we describe the ninth human DNA polymerase, Polι, encoded by the RAD30B gene. We show that human Polι violates the Watson-Crick base-pairing rule opposite template T. During base selection, human Polι preferred T-G base pairing, leading to G incorporation opposite template T. The resulting T-G base pair was less efficiently extended by human Polι compared to the Watson-Crick base pairs. Consequently, DNA synthesis frequently aborted opposite template T, a property we designated the T stop. This T stop restricted human Polι to a very short stretch of DNA synthesis. Furthermore, kinetic analyses show that human Polι copies template C with extraordinarily low fidelity, misincorporating T, A, and C with unprecedented frequencies of 1/9, 1/10, and 1/11, respectively. Human Polι incorporated one nucleotide opposite a template abasic site more efficiently than opposite a template T, suggesting a role for human Polι in DNA lesion bypass. The unique features of preferential G incorporation opposite template T and T stop suggest that DNA Polι may additionally play a specialized function in human biology.

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Protein interactions in T7 DNA replisome inhibit the bypass of abasic site by DNA polymerase

Mutagenesis ◽

10.1093/mutage/gez013 ◽

2019 ◽

Author(s):

Zhenyu Zou ◽

Tingting Liang ◽

Zhongyan Xu ◽

Jiayu Xie ◽

Shuming Zhang ◽

...

Keyword(s):

Dna Replication ◽

Dna Synthesis ◽

Dna Polymerase ◽

Protein Interactions ◽

Accessory Proteins ◽

Abasic Site ◽

Traditional Concept ◽

Translesion Dna Synthesis ◽

Dna Lesion ◽

Translesion Dna

Abstract Abasic site as a common DNA lesion blocks DNA replication and is highly mutagenic. Protein interactions in T7 DNA replisome facilitate DNA replication and translesion DNA synthesis. However, bypass of an abasic site by T7 DNA replisome has never been investigated. In this work, we used T7 DNA replisome and T7 DNA polymerase alone as two models to study DNA replication on encountering an abasic site. Relative to unmodified DNA, abasic site strongly inhibited primer extension and completely blocked strand-displacement DNA synthesis, due to the decreased fraction of enzyme–DNA productive complex and the reduced average extension rates. Moreover, abasic site at DNA fork inhibited the binding of DNA polymerase or helicase onto fork and the binding between polymerase and helicase at fork. Notably and unexpectedly, we found DNA polymerase alone bypassed an abasic site on primer/template (P/T) substrate more efficiently than did polymerase and helicase complex bypass it at fork. The presence of gp2.5 further inhibited the abasic site bypass at DNA fork. Kinetic analysis showed that this inhibition at fork relative to that on P/T was due to the decreased fraction of productive complex instead of the average extension rates. Therefore, we found that protein interactions in T7 DNA replisome inhibited the bypass of DNA lesion, different from all the traditional concept that protein interactions or accessory proteins always promote DNA replication and DNA damage bypass, providing new insights in translesion DNA synthesis performed by DNA replisome.

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Structural insights into the promutagenic bypass of the major cisplatin-induced DNA lesion

Biochemical Journal ◽

10.1042/bcj20190906 ◽

2020 ◽

Vol 477 (5) ◽

pp. 937-951

Author(s):

Hala Ouzon-Shubeita ◽

Caroline K. Vilas ◽

Seongmin Lee

Keyword(s):

Dna Polymerase ◽

Active Site ◽

Cisplatin Resistance ◽

Structural Basis ◽

Abasic Site ◽

Dna Polymerase Β ◽

Dna Lesion ◽

Human Dna ◽

Optimal Conformation ◽

Structural Insights

The cisplatin-1,2-d(GpG) (Pt-GG) intrastrand cross-link is the predominant DNA lesion generated by cisplatin. Cisplatin has been shown to predominantly induce G to T mutations and Pt-GG permits significant misincorporation of dATP by human DNA polymerase β (polβ). In agreement, polβ overexpression, which is frequently observed in cancer cells, is linked to cisplatin resistance and a mutator phenotype. However, the structural basis for the misincorporation of dATP opposite Pt-GG is unknown. Here, we report the first structures of a DNA polymerase inaccurately bypassing Pt-GG. We solved two structures of polβ misincorporating dATP opposite the 5′-dG of Pt-GG in the presence of Mg2+ or Mn2+. The Mg2+-bound structure exhibits a sub-optimal conformation for catalysis, while the Mn2+-bound structure is in a catalytically more favorable semi-closed conformation. In both structures, dATP does not form a coplanar base pairing with Pt-GG. In the polβ active site, the syn-dATP opposite Pt-GG appears to be stabilized by protein templating and pi stacking interactions, which resembles the polβ-mediated dATP incorporation opposite an abasic site. Overall, our results suggest that the templating Pt-GG in the polβ active site behaves like an abasic site, promoting the insertion of dATP in a non-instructional manner.

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Faculty Opinions recommendation of Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1029657.347531 ◽

2005 ◽

Author(s):

Errol C Friedberg

Keyword(s):

Homologous Recombination ◽

Dna Synthesis ◽

Dna Polymerase ◽

Polymerase Eta ◽

Human Dna ◽

Dna Polymerase Eta ◽

Strand Invasion

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Detection and characterization of DNA polymerase activity in Toxoplasma gondii

Parasitology ◽

10.1017/s0031182000067238 ◽

1993 ◽

Vol 107 (2) ◽

pp. 135-139 ◽

Cited By ~ 10

Author(s):

A. Makioka ◽

B. Stavros ◽

J. T. Ellis ◽

A. M. Johnson

Keyword(s):

Toxoplasma Gondii ◽

Dna Polymerase ◽

Dna Polymerases ◽

Sedimentation Coefficient ◽

Polymerase Activity ◽

Magnesium Ions ◽

Dna Polymerase Β ◽

Human Dna ◽

Approximate Molecular Weight

SUMMARYA DNA polymerase activity has been detected and characterized in crude extracts from tachzoites of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6·4 S, corresponding to an approximate molecular weight of 150000 assuming a globular shape. Like mammalian DNA polymerase α, the DNA polymerase of T. gondii was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ and ε and also cytosine-β-D-arabinofuranoside-5′-triphosphate which is an inhibitor of α polymerase. The activity was inhibited by 2′,3′-dideoxythymidine-5′-triphosphate which is an inhibitor of mammalian DNA polymerase β and γ. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 mM. The optimum potassium (K+) concentration was 50 mM and a higher concentration of K+ markedly inhibited the activity. Activity was optimal at pH 8. Monoclonal antibodies against human DNA polymerase did not bind to DNA polymerase of T. gondii. Thus the T. gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.

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Heterotrimeric PCNA Increases the Activity and Fidelity of Dbh, a Y-family Translesion DNA Polymerase Prone to Creating Single-Base Deletion Mutations

10.1101/2020.06.08.140293 ◽

2020 ◽

Author(s):

Yifeng Wu ◽

William Jaremko ◽

Ryan C. Wilson ◽

Janice D. Pata

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Polymerase Activity ◽

List Type ◽

Single Base ◽

Deletion Mutations ◽

Single Base Deletion ◽

Translesion Dna ◽

Y Family

AbstractDbh is a Y-family translesion DNA polymerase from Sulfolobus acidocaldarius, an archaeal species that grows in harsh environmental conditions. Biochemically, Dbh displays a distinctive mutational profile, creating single-base deletion mutations at extraordinarily high frequencies (up to 50%) in specific repeat sequences. In cells, however, Dbh does not appear to contribute significantly to spontaneous frameshifts in these same sequence contexts. This suggests that either the error-prone DNA synthesis activity of Dbh is reduced in vivo and/or Dbh is restricted from replicating these sequences. Here, we test the hypothesis that the propensity for Dbh to make single base deletion mutations is reduced through interaction with the S. acidocaldarius heterotrimeric sliding clamp processivity factor, PCNA-123. We first confirm that Dbh physically interacts with PCNA-123, with the interaction requiring both the PCNA-1 subunit and the C-terminal 10 amino acids of Dbh, which contain a predicted PCNA-interaction peptide (PIP) motif. This interaction stimulates the polymerase activity of Dbh, even on short, linear primer-template DNA by increasing the rate of nucleotide incorporation. This stimulation requires an intact PCNA-123 heterotrimer and a DNA duplex length of at least 18 basepairs, the minimal length predicted from structural data to bind to both the polymerase and the clamp. Finally, we find that PCNA-123 increases the fidelity of Dbh on a single-base deletion hotspot sequence 3-fold by promoting an increase in the rate of correct, but not incorrect, nucleotide addition and propose that PCNA-123 induces Dbh to adopt a more active conformation that is less prone to creating deletions during DNA synthesis.HighlightsPCNA increases the fidelity of Dbh polymerase on a deletion-hotspot sequence.The interaction stimulates incorporation of the correct, but not incorrect, nucleotide.A minimal duplex length of 18 bp is required for PCNA to stimulate polymerase activity.Structural modeling suggests that PCNA induces a conformational change in Dbh.

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Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

PLoS ONE ◽

10.1371/journal.pone.0016612 ◽

2011 ◽

Vol 6 (1) ◽

pp. e16612 ◽

Cited By ~ 18

Author(s):

Alena V. Makarova ◽

Corinn Grabow ◽

Leonid V. Gening ◽

Vyacheslav Z. Tarantul ◽

Tahir H. Tahirov ◽

...

Keyword(s):

Dna Synthesis ◽

Dna Polymerase ◽

Dna Polymerase Iota ◽

Cell Extracts ◽

Human Dna

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The fidelity of replication of the three-base-pair set adenine/thymine, hypoxanthine/cytosine and 6-thiopurine/5-methyl-2-pyrimidinone with T7 DNA polymerase

Biochemical Journal ◽

10.1042/bj20031776 ◽

2004 ◽

Vol 381 (3) ◽

pp. 709-717 ◽

Cited By ~ 12

Author(s):

Harry P. RAPPAPORT

Keyword(s):

Dna Polymerase ◽

Rate Constant ◽

Base Pair ◽

Apparent Rate Constant ◽

Polymerase Activity ◽

Exonuclease Activity ◽

Base Pairs ◽

Error Frequency ◽

Steady State Kinetics ◽

Adenine Thymine

With the goal of constructing a genetic alphabet consisting of a set of three base pairs, the fidelity of replication of the three base pairs TH (5-methyl-2-pyrimidinone)/HS (6-thiopurine; thiohypoxanthine), C/H (hypoxanthine) and T/A was evaluated using T7 DNA polymerase, a polymerase with a strong 3′→5′ exonuclease activity. An evaluation of the suitability of a new base pair for replication should include both the contribution of the fidelity of a polymerase activity and the contribution of proofreading by a 3′→5′ exonuclease activity. Using a steady-state kinetics method that included the contribution of the 3′→5′ exonuclease activity, the fidelity of replication was determined. The method determined the ratio of the apparent rate constant for the addition of a deoxynucleotide to the primer across from a template base by the polymerase activity and the rate constant for removal of the added deoxynucleotide from the primer by the 3′→5′ exonuclease activity. This ratio was designated the eni (efficiency of net incorporation). The eni of the base pair C/H was equal to or greater than the eni of T/A. The eni of the base pair TH/HS was 0.1 times that of A/T for TH in the template and 0.01 times that of A/T for HS in the template. The ratio of the eni of a mismatched deoxynucleotide to the eni of a matched deoxynucleotide was a measure of the error frequency. The error frequencies were as follows: thymine or TH opposite a template hypoxanthine, 2×10−6; HS opposite a template cytosine, <3×10−4. The remaining 24 mismatched combinations of bases gave no detectable net incorporation. Two mismatches, hypoxanthine opposite a template thymine or a template TH, showed trace incorporation in the presence of a standard dNTP complementary to the next template base. T7 DNA polymerase extended the primer beyond each of the matched base pairs of the set. The level of fidelity of replication of the three base pairs with T7 DNA polymerase suggests that they are adequate for a three-base-pair alphabet for DNA replication.

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Error-prone bypass of certain DNA lesions by the human DNA polymerase κ

Genes & Development ◽

10.1101/gad.14.13.1589 ◽

2000 ◽

Vol 14 (13) ◽

pp. 1589-1594 ◽

Cited By ~ 3

Author(s):

Eiji Ohashi ◽

Tomoo Ogi ◽

Rika Kusumoto ◽

Shigenori Iwai ◽

Chikahide Masutani ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Polymerase ◽

High Frequency ◽

Dna Lesions ◽

Human Homolog ◽

Abasic Site ◽

Thymine Dimer ◽

Human Dna

The Escherichia coli protein DinB is a newly identified error-prone DNA polymerase. Recently, a human homolog of DinB was identified and named DINB1. We report that the DINB1gene encodes a DNA polymerase (designated polκ), which incorporates mismatched bases on a nondamaged template with a high frequency. Moreover, polκ bypasses an abasic site andN-2–acetylaminofluorene (AAF)-adduct in an error-prone manner but does not bypass a cis–syn or (6-4) thymine–thymine dimer or a cisplatin-adduct. Therefore, our results implicate an important role for polκ in the mutagenic bypass of certain types of DNA lesions.

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