scholarly journals The stabilized Pol31–Pol3 interface counteracts Pol32 ablation with differential effects on repair

2021 ◽  
Vol 4 (9) ◽  
pp. e202101138
Author(s):  
Kenji Shimada ◽  
Monika Tsai-Pflugfelder ◽  
Niloofar Davoodi Vijeh Motlagh ◽  
Neda Delgoshaie ◽  
Jeannette Fuchs ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Methyl Methanesulfonate ◽  
Replication Forks ◽  
Differential Effects ◽  
Protein Levels ◽  
Dna Replication And Repair ◽  
Interaction Domains ◽  
Repair Pathways ◽  
Break Induced Replication

DNA polymerase δ, which contains the catalytic subunit, Pol3, Pol31, and Pol32, contributes both to DNA replication and repair. The deletion of pol31 is lethal, and compromising the Pol3–Pol31 interaction domains confers hypersensitivity to cold, hydroxyurea (HU), and methyl methanesulfonate, phenocopying pol32Δ. We have identified alanine-substitutions in pol31 that suppress these deficiencies in pol32Δ cells. We characterize two mutants, pol31-T415A and pol31-W417A, which map to a solvent-exposed loop that mediates Pol31–Pol3 and Pol31–Rev3 interactions. The pol31-T415A substitution compromises binding to the Pol3 CysB domain, whereas Pol31-W417A improves it. Importantly, loss of Pol32, such as pol31-T415A, leads to reduced Pol3 and Pol31 protein levels, which are restored by pol31-W417A. The mutations have differential effects on recovery from acute HU, break-induced replication and trans-lesion synthesis repair pathways. Unlike trans-lesion synthesis and growth on HU, the loss of break-induced replication in pol32Δ cells is not restored by pol31-W417A, highlighting pathway-specific roles for Pol32 in fork-related repair. Intriguingly, CHIP analyses of replication forks on HU showed that pol32Δ and pol31-T415A indirectly destabilize DNA pol α and pol ε at stalled forks.

scholarly journals DNA polymerase ζ in DNA replication and repair

2019 ◽  
Vol 47 (16) ◽  
pp. 8348-8361 ◽  
Author(s):  
Sara K Martin ◽  
Richard D Wood
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Translesion Synthesis ◽  
Dna Lesions ◽  
Replication Forks ◽  
Dna Replication And Repair ◽  
Radiation Induced ◽  
Break Induced Replication

Abstract Here, we survey the diverse functions of DNA polymerase ζ (pol ζ) in eukaryotes. In mammalian cells, REV3L (3130 residues) is the largest catalytic subunit of the DNA polymerases. The orthologous subunit in yeast is Rev3p. Pol ζ also includes REV7 subunits (encoded by Rev7 in yeast and MAD2L2 in mammalian cells) and two subunits shared with the replicative DNA polymerase, pol δ. Pol ζ is used in response to circumstances that stall DNA replication forks in both yeast and mammalian cells. The best-examined situation is translesion synthesis at sites of covalent DNA lesions such as UV radiation-induced photoproducts. We also highlight recent evidence that uncovers various roles of pol ζ that extend beyond translesion synthesis. For instance, pol ζ is also employed when the replisome operates sub-optimally or at difficult-to-replicate DNA sequences. Pol ζ also participates in repair by microhomology mediated break-induced replication. A rev3 deletion is tolerated in yeast but Rev3l disruption results in embryonic lethality in mice. Inactivation of mammalian Rev3l results in genomic instability and invokes cell death and senescence programs. Targeting of pol ζ function may be a useful strategy in cancer therapy, although chromosomal instability associated with pol ζ deficiency must be considered.

scholarly journals Escherichia coli Cells with Increased Levels of DnaA and Deficient in Recombinational Repair Have Decreased Viability

2003 ◽  
Vol 185 (2) ◽  
pp. 630-644 ◽  
Author(s):  
Aline V. Grigorian ◽  
Rachel B. Lustig ◽  
Elena C. Guzmán ◽  
Joseph M. Mahaffy ◽  
Judith W. Zyskind
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Replication Fork ◽  
Replication Initiation ◽  
Recombinational Repair ◽  
Replication Forks ◽  
Dna Replication Initiation ◽  
Escherichia Coli Cells ◽  
Repair Pathways ◽  
Stalled Replication Forks

ABSTRACT The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, β clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, β clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the β clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 μm) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional ∼100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.

scholarly journals Break-Induced Replication Repair of Damaged Forks Induces Genomic Duplications in Human Cells

Science ◽  
2013 ◽  
Vol 343 (6166) ◽  
pp. 88-91 ◽  
Author(s):  
Lorenzo Costantino ◽  
Sotirios K. Sotiriou ◽  
Juha K. Rantala ◽  
Simon Magin ◽  
Emil Mladenov ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cell Cycle Progression ◽  
Cyclin E ◽  
Replication Stress ◽  
Replication Forks ◽  
Dna Polymerase Delta ◽  
Dna Double Strand Breaks ◽  
Dna Replication Stress ◽  
Recombination Pathway ◽  
Break Induced Replication

In budding yeast, one-ended DNA double-strand breaks (DSBs) and damaged replication forks are repaired by break-induced replication (BIR), a homologous recombination pathway that requires the Pol32 subunit of DNA polymerase delta. DNA replication stress is prevalent in cancer, but BIR has not been characterized in mammals. In a cyclin E overexpression model of DNA replication stress, POLD3, the human ortholog of POL32, was required for cell cycle progression and processive DNA synthesis. Segmental genomic duplications induced by cyclin E overexpression were also dependent on POLD3, as were BIR-mediated recombination events captured with a specialized DSB repair assay. We propose that BIR repairs damaged replication forks in mammals, accounting for the high frequency of genomic duplications in human cancers.

Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair

Nature ◽  
1994 ◽  
Vol 371 (6497) ◽  
pp. 534-537 ◽  
Author(s):  
Rong Li ◽  
Shou Waga ◽  
Gregory J. Hannon ◽  
David Beach ◽  
Bruce Stillman
Keyword(s):  
Dna Replication ◽  
Cdk Inhibitor ◽  
Differential Effects ◽  
Dna Replication And Repair

scholarly journals Serotype-Specific Reorganization of the Mre11 Complex by Adenoviral E4orf3 Proteins

2005 ◽  
Vol 79 (11) ◽  
pp. 6664-6673 ◽  
Author(s):  
Travis H. Stracker ◽  
Darwin V. Lee ◽  
Christian T. Carson ◽  
Felipe D. Araujo ◽  
David A. Ornelles ◽  
...  
Keyword(s):  
Dna Replication ◽  
Protein Production ◽  
Adenovirus Type ◽  
Expression Vectors ◽  
Gene Products ◽  
Late Protein ◽  
Mre11 Complex ◽  
Dna Replication And Repair ◽  
Repair Pathways ◽  
Adenovirus Type 5

ABSTRACT The early transcriptional region 4 (E4) of adenovirus type 5 (Ad5) encodes gene products that modulate splicing, apoptosis, transcription, DNA replication, and repair pathways. Viruses lacking both E4orf3 and E4orf6 have a severe replication defect, partially characterized by the formation of genome concatemers. Concatemer formation is dependent upon the cellular Mre11 complex and is prevented by both the E4orf3 and E4orf6 proteins. The Mre11/Rad50/Nbs1 proteins are targeted for proteasome-mediated degradation by the Ad5 viral E1b55K/E4orf6 complex. The expression of Ad5 E4orf3 causes a redistribution of Mre11 complex members and results in their exclusion from viral replication centers. For this study, we further analyzed the interactions of E4 proteins from different adenovirus serotypes with the Mre11 complex. Analyses of infections with serotypes Ad4 and Ad12 demonstrated that the degradation of Mre11/Rad50/Nbs1 proteins is a conserved feature of the E1b55K/E4orf6 complex. Surprisingly, Nbs1 and Rad50 were localized to the replication centers of both Ad4 and Ad12 viruses prior to Mre11 complex degradation. The transfection of expression vectors for the E4orf3 proteins of Ad4 and Ad12 did not alter the localization of Mre11 complex members. The E4orf3 proteins of Ad4 and Ad12 also failed to complement defects in both concatemer formation and late protein production of a virus with a deletion of E4. These results reveal surprising differences among the highly conserved E4orf3 proteins from different serotypes in the ability to disrupt the Mre11 complex.

scholarly journals DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions

2012 ◽  
Vol 183 (2) ◽  
pp. 122-131 ◽  
Author(s):  
Bruno Luiz Fonseca Schamber-Reis ◽  
Sheila Nardelli ◽  
Carlos Gustavo Régis-Silva ◽  
Priscila Carneiro Campos ◽  
Paula Gonçalves Cerqueira ◽  
...  
Keyword(s):  
Dna Replication ◽  
Trypanosoma Cruzi ◽  
Dna Polymerase ◽  
Dna Polymerase Beta ◽  
Kinetoplast Dna ◽  
Polymerase Beta ◽  
Dna Replication And Repair

scholarly journals DNA polymerase IV primarily operates outside of DNA replication forks in Escherichia coli

PLoS Genetics ◽  
2018 ◽  
Vol 14 (1) ◽  
pp. e1007161 ◽  
Author(s):  
Sarah S. Henrikus ◽  
Elizabeth A. Wood ◽  
John P. McDonald ◽  
Michael M. Cox ◽  
Roger Woodgate ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Replication Forks ◽  
Dna Polymerase Iv

scholarly journals Haloferax volcanii —a model archaeon for studying DNA replication and repair

Open Biology ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 200293
Author(s):  
Patricia Pérez-Arnaiz ◽  
Ambika Dattani ◽  
Victoria Smith ◽  
Thorsten Allers
Keyword(s):  
Dna Replication ◽  
Genetic Manipulation ◽  
Haloferax Volcanii ◽  
Cellular Biology ◽  
Phenotypic Screening ◽  
The Third ◽  
Dna Replication And Repair ◽  
Repair Pathways ◽  
Halophilic Species ◽  
The Relationship

The tree of life shows the relationship between all organisms based on their common ancestry. Until 1977, it comprised two major branches: prokaryotes and eukaryotes. Work by Carl Woese and other microbiologists led to the recategorization of prokaryotes and the proposal of three primary domains: Eukarya, Bacteria and Archaea. Microbiological, genetic and biochemical techniques were then needed to study the third domain of life. Haloferax volcanii , a halophilic species belonging to the phylum Euryarchaeota, has provided many useful tools to study Archaea, including easy culturing methods, genetic manipulation and phenotypic screening. This review will focus on DNA replication and DNA repair pathways in H. volcanii , how this work has advanced our knowledge of archaeal cellular biology, and how it may deepen our understanding of bacterial and eukaryotic processes.

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