31 research outputs found
Mismatch RepairβIndependent Increase in Spontaneous Mutagenesis in Yeast Lacking Non-Essential Subunits of DNA Polymerase Ξ΅
Yeast DNA polymerase Ξ΅ (Pol Ξ΅) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol Ξ΅ also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol Ξ΅ in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol Ξ΅ holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol Ξ΅ proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Ξdpb4Ξ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Ξdpb4Ξ strains is partly dependent on REV3, as well as the proofreading capacity of Pol Ξ΄. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol Ξ΅ and the template DNA during processive DNA synthesis and during processive 3β² to 5β²exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ΞΆ or Pol Ξ΄, in a post-replication process that generates errors not corrected by the DNA mismatch repair system
Birth characteristics and the risk of childhood rhabdomyosarcoma based on histological subtype
Overexpression of DNA Polymerase Zeta Reduces the Mitochondrial Mutability Caused by Pathological Mutations in DNA Polymerase Gamma in Yeast
In yeast, DNA polymerase zeta (Rev3 and Rev7) and Rev1, involved in the error-prone translesion synthesis during replication of nuclear DNA, localize also in mitochondria. We show that overexpression of Rev3 reduced the mtDNA extended mutability caused by a subclass of pathological mutations in Mip1, the yeast mitochondrial DNA polymerase orthologous to human Pol gamma. This beneficial effect was synergistic with the effect achieved by increasing the dNTPs pools. Since overexpression of Rev3 is detrimental for nuclear DNA mutability, we constructed a mutant Rev3 isoform unable to migrate into the nucleus: its overexpression reduced mtDNA mutability without increasing the nuclear one
Positive parenting for healthy living (Triple P) for parents of children with type 1 diabetes: protocol of a randomised controlled trial
Factors affecting newly increased construction land at different development stages: Evidence from 352 Chinese cities
The importance of an interaction network for proper DNA polymerase ΞΆ heterotetramer activity
Characterization of Tachykinin Receptors by Ligand Binding Studies and by Utilization of Conformationally Restricted Tachykinin Analogues
An exploration of the relationship between families of deceased organ donors and transplant recipients: A systematic review and qualitative synthesis
Hypermutability of Damaged Single-Strand DNA Formed at Double-Strand Breaks and Uncapped Telomeres in Yeast Saccharomyces cerevisiae
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purposeDOI: 10.1371/journal.pgen.1000264The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase f. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UVinduced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution