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 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.

scholarly journals Pif1-family helicases cooperate to suppress widespread replication fork arrest at tRNA genes

2016 ◽  
Author(s):  
Joseph S. Osmundson ◽  
Jayashree Kumar ◽  
Rani Yeung ◽  
Duncan J. Smith
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Trna Genes ◽  
Strand Displacement ◽  
Dna Polymerase Δ ◽  
Replication Fork Arrest ◽  
Nuclear Processes ◽  
Lagging Strand

ABSTRACTSaccharomyces cerevisiaeencodes two distinct Pif1-family helicases – Pif1 and Rrm3 – which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterize the roles of Pif1 helicases in replisome progression and lagging-strand synthesis inS. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulate strand-displacement by DNA polymerase δ during lagging-strand synthesis. By analyzing replisome mobility inpif1andrrm3mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes; consistent with this observation, we find that head-on collisions between tRNA transcription and replisome progression are under-represented in theS. cerevisiaegenome. Further, we demonstrate that tRNA-mediated arrest is R-loop independent, and propose that replisome arrest and DNA damage are mechanistically separable.

scholarly journals Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis

2007 ◽  
Vol 27 (21) ◽  
pp. 7669-7682 ◽  
Author(s):  
Ranga N. Venkatesan ◽  
Piper M. Treuting ◽  
Evan D. Fuller ◽  
Robert E. Goldsby ◽  
Thomas H. Norwood ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Dna Polymerase ◽  
Active Site ◽  
Genetic Recombination ◽  
Human Cancer ◽  
Disease Incidence ◽  
Spontaneous Mutation ◽  
Heterozygous Mutation ◽  
Wild Type ◽  
Dna Polymerase Δ

ABSTRACT Mammalian DNA polymerase δ (Pol δ) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol δ. Homozygous Pold1 L604G/L604G and Pold1 L604K/L604K mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1 +/L604G mice did not differ, while that of Pold1 +/L604K mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1 +/L604K mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase δ reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer.

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