scholarly journals MTH1 inhibitor TH588 induces mitosis-dependent accumulation of genomic 8-oxodG and disturbs mitotic progression

2019 ◽  
Author(s):  
Sean G Rudd ◽  
Helge Gad ◽  
Nuno Amaral ◽  
Anna Hagenkort ◽  
Petra Groth ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Replication Fork ◽  
Dna Polymerases ◽  
Mitotic Cell ◽  
Mitotic Arrest ◽  
Oxygen Species ◽  
Mitotic Progression ◽  
Dna Polymerase Delta ◽  
Replication Fork Progression ◽  
In Cells

ABSTRACTReactive oxygen species (ROS) oxidise nucleotide triphosphate pools (e.g., 8-oxodGTP), which may kill cells if incorporated into DNA. Whether cancers avoid poisoning from oxidised nucleotides by preventing incorporation via the oxidised purine diphosphatase MTH1 remains under debate. Also, little is known about DNA polymerases incorporating oxidised nucleotides in cells or how oxidised nucleotides in DNA become toxic. We show replacement of one of the main DNA replicases in human cells, DNA polymerase delta (Pol δ), to an error-prone variant allows increased 8-oxodG accumulation into DNA following treatment with the MTH1 inhibitor (MTH1i) TH588. The resulting elevated genomic 8-oxodG correlates with increased cytotoxicity of TH588. Interestingly, no substantial perturbation of replication fork progression is observed, but rather mitotic progression is impaired and mitotic DNA synthesis triggered. Reducing mitotic arrest by reversin treatment prevents accumulation of genomic 8-oxodG and reduces cytotoxicity of TH588, in line with the notion that mitotic arrest is required for ROS build-up and oxidation of the nucleotide pool. Furthermore, we demonstrate delayed mitosis and increased mitotic cell death following TH588 treatment in cells expressing the error-prone Pol δ variant, which is not observed following treatments with anti-mitotic agents, thus linking incorporation of oxidised nucleotides and disturbed mitotic progression.

Primary structure of the catalytic subunit of calf thymus DNA polymerase .delta.: sequence similarities with other DNA polymerases

Biochemistry ◽  
1991 ◽  
Vol 30 (51) ◽  
pp. 11742-11750 ◽  
Author(s):  
Jian Zhang ◽  
Dominic W. Chung ◽  
Cheng Keat Tan ◽  
Kathleen M. Downey ◽  
Earl W. Davie ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Primary Structure ◽  
Dna Polymerases ◽  
Catalytic Subunit ◽  
Calf Thymus Dna ◽  
Dna Polymerase Delta ◽  
Calf Thymus ◽  
Delta Sequence ◽  
Sequence Similarities

scholarly journals Active Site Mutations in Mammalian DNA Polymerase δ Alter Accuracy and Replication Fork Progression

2010 ◽  
Vol 285 (42) ◽  
pp. 32264-32272 ◽  
Author(s):  
Michael W. Schmitt ◽  
Ranga N. Venkatesan ◽  
Marie-Jeanne Pillaire ◽  
Jean-Sébastien Hoffmann ◽  
Julia M. Sidorova ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Replication Fork ◽  
Dna Polymerase Δ ◽  
Replication Fork Progression ◽  
Active Site Mutations

scholarly journals DNA polymerases required for repair of UV-induced damage in Saccharomyces cerevisiae.

1995 ◽  
Vol 15 (4) ◽  
pp. 2173-2179 ◽  
Author(s):  
M E Budd ◽  
J L Campbell
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Polymerase Delta ◽  
Weight Changes ◽  
Repair Synthesis ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Quadruple Mutant ◽  
Cellular Dna ◽  
Repair Defect

The ability of yeast DNA polymerase mutant strains to carry out repair synthesis after UV irradiation was studied by analysis of postirradiation molecular weight changes in cellular DNA. Neither DNA polymerase alpha, delta, epsilon, nor Rev3 single mutants evidenced a defect in repair. A mutant defective in all four of these DNA polymerases, however, showed accumulation of single-strand breaks, indicating defective repair. Pairwise combination of polymerase mutations revealed a repair defect only in DNA polymerase delta and epsilon double mutants. The extent of repair in the double mutant was no greater than that in the quadruple mutant, suggesting that DNA polymerases alpha and Rev3p play very minor, if any, roles. Taken together, the data suggest that DNA polymerases delta and epsilon are both potentially able to perform repair synthesis and that in the absence of one, the other can efficiently substitute. Thus, two of the DNA polymerases involved in DNA replication are also involved in DNA repair, adding to the accumulating evidence that the two processes are coupled.

scholarly journals Mutagenesis mechanism of the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine

2020 ◽  
Vol 48 (9) ◽  
pp. 5119-5134 ◽  
Author(s):  
Myong-Chul Koag ◽  
Hunmin Jung ◽  
Seongmin Lee
Keyword(s):  
Reactive Oxygen Species ◽  
Crystal Structures ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Reactive Oxygen ◽  
Minor Groove ◽  
Oxygen Species ◽  
Base Pairing ◽  
Human Dna ◽  
Hoogsteen Base Pairing

Abstract Reactive oxygen species generate the genotoxic 8-oxoguanine (oxoG) and 8-oxoadenine (oxoA) as major oxidative lesions. The mutagenicity of oxoG is attributed to the lesion's ability to evade the geometric discrimination of DNA polymerases by adopting Hoogsteen base pairing with adenine in a Watson–Crick-like geometry. Compared with oxoG, the mutagenesis mechanism of oxoA, which preferentially induces A-to-C mutations, is poorly understood. In the absence of protein contacts, oxoA:G forms a wobble conformation, the formation of which is suppressed in the catalytic site of most DNA polymerases. Interestingly, human DNA polymerase η (polη) proficiently incorporates dGTP opposite oxoA, suggesting the nascent oxoA:dGTP overcomes the geometric discrimination of polη. To gain insights into oxoA-mediated mutagenesis, we determined crystal structures of polη bypassing oxoA. When paired with dGTP, oxoA adopted a syn-conformation and formed Hoogsteen pairing while in a wobble geometry, which was stabilized by Gln38-mediated minor groove contacts to oxoA:dGTP. Gln38Ala mutation reduced misinsertion efficiency ∼55-fold, indicating oxoA:dGTP misincorporation was promoted by minor groove interactions. Also, the efficiency of oxoA:dGTP insertion by the X-family polβ decreased ∼380-fold when Asn279-mediated minor groove contact to dGTP was abolished. Overall, these results suggest that, unlike oxoG, oxoA-mediated mutagenesis is greatly induced by minor groove interactions.

scholarly journals DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (1) ◽  
pp. 496-505 ◽  
Author(s):  
M E Budd ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nonpermissive Temperature ◽  
Dna Polymerase Delta ◽  
Dna Polymerase Epsilon ◽  
Dna Polymerase Alpha ◽  
Polymerase Epsilon

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.

scholarly journals DNA polymerases delta and epsilon are required for chromosomal replication in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (1) ◽  
pp. 496-505
Author(s):  
M E Budd ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nonpermissive Temperature ◽  
Dna Polymerase Delta ◽  
Dna Polymerase Epsilon ◽  
Dna Polymerase Alpha ◽  
Polymerase Epsilon

Three DNA polymerases, alpha, delta, and epsilon are required for viability in Saccharomyces cerevisiae. We have investigated whether DNA polymerases epsilon and delta are required for DNA replication. Two temperature-sensitive mutations in the POL2 gene, encoding DNA polymerase epsilon, have been identified by using the plasmid shuffle technique. Alkaline sucrose gradient analysis of DNA synthesis products in the mutant strains shows that no chromosomal-size DNA is formed after shift of an asynchronous culture to the nonpermissive temperature. The only DNA synthesis observed is a reduced quantity of short DNA fragments. The DNA profiles of replication intermediates from these mutants are similar to those observed with DNA synthesized in mutants deficient in DNA polymerase alpha under the same conditions. The finding that DNA replication stops upon shift to the nonpermissive temperature in both DNA polymerase alpha- and DNA polymerase epsilon- deficient strains shows that both DNA polymerases are involved in elongation. By contrast, previous studies on pol3 mutants, deficient in DNA polymerase delta, suggested that there was considerable residual DNA synthesis at the nonpermissive temperature. We have reinvestigated the nature of DNA synthesis in pol3 mutants. We find that pol3 strains are defective in the synthesis of chromosomal-size DNA at the restrictive temperature after release from a hydroxyurea block. These results demonstrate that yeast DNA polymerase delta is also required at the replication fork.

scholarly journals Y-family DNA polymerases respond to DNA damage-independent inhibition of replication fork progression

2006 ◽  
Vol 25 (4) ◽  
pp. 868-879 ◽  
Author(s):  
Veronica G Godoy ◽  
Daniel F Jarosz ◽  
Fabianne L Walker ◽  
Lyle A Simmons ◽  
Graham C Walker
Keyword(s):  
Dna Damage ◽  
Replication Fork ◽  
Dna Polymerases ◽  
Replication Fork Progression ◽  
Y Family

scholarly journals Alternative Mechanisms for Coordinating Polymerase α and MCM Helicase

2009 ◽  
Vol 30 (2) ◽  
pp. 423-435 ◽  
Author(s):  
Chanmi Lee ◽  
Ivan Liachko ◽  
Roxane Bouten ◽  
Zvi Kelman ◽  
Bik K. Tye
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Molecular Motors ◽  
Replication Fork ◽  
Dna Polymerases ◽  
Physical Stability ◽  
Dependent Manner ◽  
Replication Forks ◽  
Mcm Helicase ◽  
The Stability

ABSTRACT Functional coordination between DNA replication helicases and DNA polymerases at replication forks, achieved through physical linkages, has been demonstrated in prokaryotes but not in eukaryotes. In Saccharomyces cerevisiae, we showed that mutations that compromise the activity of the MCM helicase enhance the physical stability of DNA polymerase α in the absence of their presumed linker, Mcm10. Mcm10 is an essential DNA replication protein implicated in the stable assembly of the replisome by virtue of its interaction with the MCM2-7 helicase and Polα. Dominant mcm2 suppressors of mcm10 mutants restore viability by restoring the stability of Polα without restoring the stability of Mcm10, in a Mec1-dependent manner. In this process, the single-stranded DNA accumulation observed in the mcm10 mutant is suppressed. The activities of key checkpoint regulators known to be important for replication fork stabilization contribute to the efficiency of suppression. These results suggest that Mcm10 plays two important roles as a linker of the MCM helicase and Polα at the elongating replication fork—first, to coordinate the activities of these two molecular motors, and second, to ensure their physical stability and the integrity of the replication fork.

scholarly journals A Hypothesis for DNA Viruses as the Origin of Eukaryotic Replication Proteins

2000 ◽  
Vol 74 (15) ◽  
pp. 7079-7084 ◽  
Author(s):  
Luis P. Villarreal ◽  
Victor R. DeFilippis
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Reverse Direction ◽  
Amino Terminus ◽  
Replication Proteins ◽  
Dna Polymerase Delta ◽  
Dna Virus ◽  
Significant Similarity ◽  
Dna Viruses ◽  
Eukaryotic Dna

ABSTRACT The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction.

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