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

Yeast ◽  
2020 ◽  
Author(s):  
Matthew J. Young ◽  
Robin J. Imperial ◽  
Suman Lakhi ◽  
Deborah A. Court
Keyword(s):  
Mitochondrial Dna ◽  
Enzyme Activity ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Polymerase Domain ◽  
Mitochondrial Dna Polymerase

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

Mitochondrion ◽  
2021 ◽  
Author(s):  
Elena J. Ciesielska ◽  
Shalom Kim ◽  
Hyacintha-ghislaine M. Bisimwa ◽  
Cody Grier ◽  
Md. Mostafijur Rahman ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Mitochondrial Dna Polymerase

scholarly journals Properties of mitochondrial DNA polymerase in mitochondrial DNA synthesis in yeast.

1995 ◽  
Vol 42 (3) ◽  
pp. 317-324 ◽  
Author(s):  
T K Biswas ◽  
P Sengupta ◽  
R Green ◽  
P Hakim ◽  
B Biswas ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mt Dna ◽  
Double Stranded Dna ◽  
Dna Template ◽  
Mitochondrial Dna Polymerase

Mitochondrial DNA polymerase from Saccharomyces cerevisiae, purified 3500 fold, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into three polypeptides. The major 150 kDa polypeptide was probably the catalytic subunit of the mitochondrial (mt) DNA polymerase and the other two polypeptides could be either proteolytic cleavage products of the polymerase, other subunits of the enzyme or protein contaminants. The mtDNA polymerase preferred an A+T-rich DNA template and did not require any RNA primer for DNA synthesis, at least under in vitro reaction conditions. It showed higher processivity on a double-stranded linear DNA template than on a single-stranded circular DNA template, and was capable of synthesizing at least about 1200 nucleotide primer-extended products without any major pause on a double-stranded DNA template.

scholarly journals Mismatch-specific 3'----5' exonuclease associated with the mitochondrial DNA polymerase from Drosophila embryos

1989 ◽  
Vol 86 (17) ◽  
pp. 6469-6473 ◽  
Author(s):  
L S Kaguni ◽  
M W Olson
Keyword(s):  
Mitochondrial Dna ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Templates ◽  
Polymerase Gamma ◽  
Dna Polymerase Gamma ◽  
Mitochondrial Dna Replication ◽  
Mitochondrial Dna Polymerase ◽  
Hydrolysis Of ◽  
Drosophila Embryos

The mitochondrial DNA polymerase from Drosophila embryos lacks dNTP turnover activity. However, a potent 3'----5' exonuclease activity can be detected by a specific assay in which the exonuclease excises mispaired nucleotides at the 3' termini of primed synthetic and natural DNA templates. The excision of a mispaired nucleotide occurs at a significantly greater rate than excision of a correctly paired nucleotide and, under conditions of DNA synthesis, hydrolysis of a mispaired terminal nucleotide occurs prior to primer extension. The 3'----5' exonuclease copurifies quantitatively with DNA polymerase gamma and cosediments with the nearly homogeneous enzyme under native conditions. These results suggest that the 3'----5' exonuclease provides a proofreading function to enhance the fidelity of DNA synthesis during Drosophila mitochondrial DNA replication.

scholarly journals Translesion Synthesis Past Acrolein-derived DNA Adducts by Human Mitochondrial DNA Polymerase γ

2013 ◽  
Vol 288 (20) ◽  
pp. 14247-14255 ◽  
Author(s):  
Rajesh Kasiviswanathan ◽  
Irina G. Minko ◽  
R. Stephen Lloyd ◽  
William C. Copeland
Keyword(s):  
Mitochondrial Dna ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Adducts ◽  
Translesion Synthesis ◽  
Minor Groove ◽  
Purine Nucleotides ◽  
Nucleotide Incorporation ◽  
Mitochondrial Dna Polymerase ◽  
Following Error

Acrolein, a mutagenic aldehyde, is produced endogenously by lipid peroxidation and exogenously by combustion of organic materials, including tobacco products. Acrolein reacts with DNA bases forming exocyclic DNA adducts, such as γ-hydroxy-1,N2-propano-2′-deoxyguanosine (γ-HOPdG) and γ-hydroxy-1,N6-propano-2′-deoxyadenosine (γ-HOPdA). The bulky γ-HOPdG adduct blocks DNA synthesis by replicative polymerases but can be bypassed by translesion synthesis polymerases in the nucleus. Although acrolein-induced adducts are likely to be formed and persist in mitochondrial DNA, animal cell mitochondria lack specialized translesion DNA synthesis polymerases to tolerate these lesions. Thus, it is important to understand how pol γ, the sole mitochondrial DNA polymerase in human cells, acts on acrolein-adducted DNA. To address this question, we investigated the ability of pol γ to bypass the minor groove γ-HOPdG and major groove γ-HOPdA adducts using single nucleotide incorporation and primer extension analyses. The efficiency of pol γ-catalyzed bypass of γ-HOPdG was low, and surprisingly, pol γ preferred to incorporate purine nucleotides opposite the adduct. Pol γ also exhibited ∼2-fold lower rates of excision of the misincorporated purine nucleotides opposite γ-HOPdG compared with the corresponding nucleotides opposite dG. Extension of primers from the termini opposite γ-HOPdG was accomplished only following error-prone purine nucleotide incorporation. However, pol γ preferentially incorporated dT opposite the γ-HOPdA adduct and efficiently extended primers from the correctly paired terminus, indicating that γ-HOPdA is probably nonmutagenic. In summary, our data suggest that acrolein-induced exocyclic DNA lesions can be bypassed by mitochondrial DNA polymerase but, in the case of the minor groove γ-HOPdG adduct, at the cost of unprecedented high mutation rates.

scholarly journals The tamas Gene, Identified as a Mutation That Disrupts Larval Behavior in Drosophila melanogaster, Codes for the Mitochondrial DNA Polymerase Catalytic Subunit (DNApol-γ125)

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1809-1824 ◽  
Author(s):  
Balaji Iyengar ◽  
John Roote ◽  
Ana Regina Campos
Keyword(s):  
Mitochondrial Dna ◽  
Drosophila Melanogaster ◽  
Mutant Strain ◽  
Dna Polymerase ◽  
Complementation Group ◽  
Behavioral Changes ◽  
Catalytic Subunit ◽  
Primary Deficit ◽  
Mitochondrial Dna Polymerase ◽  
Food Substrate

AbstractFrom a screen of pupal lethal lines of Drosophila melanogaster we identified a mutant strain that displayed a reproducible reduction in the larval response to light. Moreover, this mutant strain showed defects in the development of the adult visual system and failure to undergo behavioral changes characteristic of the wandering stage. The foraging third instar larvae remained in the food substrate for a prolonged period and died at or just before pupariation. Using a new assay for individual larval photobehavior we determined that the lack of response to light in these mutants was due to a primary deficit in locomotion. The mutation responsible for these phenotypes was mapped to the lethal complementation group l(2)34Dc, which we renamed tamas (translated from Sanskrit as “dark inertia”). Sequencing of mutant alleles demonstrated that tamas codes for the mitochondrial DNA polymerase catalytic subunit (DNApol-γ125).

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