scholarly journals Regulation of Gross Chromosomal Rearrangements by Ubiquitin and SUMO Ligases in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (4) ◽  
pp. 1424-1433 ◽  
Author(s):  
Akira Motegi ◽  
Karen Kuntz ◽  
Anju Majeed ◽  
Stephanie Smith ◽  
Kyungjae Myung
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
De Novo ◽  
Chromosomal Rearrangements ◽  
Nuclear Antigen ◽  
Replication Forks ◽  
Gross Chromosomal Rearrangements ◽  
Sumo Ligase ◽  
Free Mode ◽  
Proliferating Cell

ABSTRACT Gross chromosomal rearrangements (GCRs) are frequently observed in many cancers. Previously, we showed that inactivation of Rad5 or Rad18, ubiquitin ligases (E3) targeting for proliferating cell nuclear antigen (PCNA), increases the de novo telomere addition type of GCR (S. Smith, J. Y. Hwang, S. Banerjee, A. Majeed, A. Gupta, and K. Myung, Proc. Natl. Acad. Sci. USA 101:9039-9044, 2004). GCR suppression by Rad5 and Rad18 appears to be exerted by the RAD5-dependent error-free mode of bypass DNA repair. In contrast, Siz1 SUMO ligase and another ubiquitin ligase, Bre1, which target for PCNA and histone H2B, respectively, have GCR-supporting activities. Inactivation of homologous recombination (HR) proteins or the helicase Srs2 reduces GCR rates elevated by the rad5 or rad18 mutation. GCRs are therefore likely to be produced through the restrained recruitment of an HR pathway to stalled DNA replication forks. Since this HR pathway is compatible with Srs2, it is not a conventional form of recombinational pathway. Lastly, we demonstrate that selection of proper DNA repair pathways to stalled DNA replication forks is controlled by the Mec1-dependent checkpoint and is executed by cooperative functions of Siz1 and Srs2. We propose a mechanism for how defects in these proteins could lead to diverse outcomes (proper repair or GCR formation) through different regulation of DNA repair machinery.

scholarly journals The Saccharomyces cerevisiae Rad6 Postreplication Repair and Siz1/Srs2 Homologous Recombination-Inhibiting Pathways Process DNA Damage That Arises in asf1 Mutants

2009 ◽  
Vol 29 (19) ◽  
pp. 5226-5237 ◽  
Author(s):  
Ellen S. Kats ◽  
Jorrit M. Enserink ◽  
Sandra Martinez ◽  
Richard D. Kolodner
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Chromosomal Rearrangements ◽  
Nuclear Antigen ◽  
Recombination Suppression ◽  
Postreplication Repair ◽  
Gross Chromosomal Rearrangements ◽  
Translesion Bypass ◽  
The Relationship ◽  
Proliferating Cell

ABSTRACT The Asf1 and Rad6 pathways have been implicated in a number of common processes such as suppression of gross chromosomal rearrangements (GCRs), DNA repair, modification of chromatin, and proper checkpoint functions. We examined the relationship between Asf1 and different gene products implicated in postreplication repair (PRR) pathways in the suppression of GCRs, checkpoint function, sensitivity to hydroxyurea (HU) and methyl methanesulfonate (MMS), and ubiquitination of proliferating cell nuclear antigen (PCNA). We found that defects in Rad6 PRR pathway and Siz1/Srs2 homologous recombination suppression (HRS) pathway genes suppressed the increased GCR rates seen in asf1 mutants, which was independent of translesion bypass polymerases but showed an increased dependency on Dun1. Combining an asf1 deletion with different PRR mutations resulted in a synergistic increase in sensitivity to chronic HU and MMS treatment; however, these double mutants were not checkpoint defective, since they were capable of recovering from acute treatment with HU. Interestingly, we found that Asf1 and Rad6 cooperate in ubiquitination of PCNA, indicating that Rad6 and Asf1 function in parallel pathways that ubiquitinate PCNA. Our results show that ASF1 probably contributes to the maintenance of genome stability through multiple mechanisms, some of which involve the PRR and HRS pathways.

scholarly journals Pivotal roles of PCNA loading and unloading in heterochromatin function

2018 ◽  
Vol 115 (9) ◽  
pp. E2030-E2039 ◽  
Author(s):  
Ryan Janke ◽  
Grant A. King ◽  
Martin Kupiec ◽  
Jasper Rine
Keyword(s):  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Transcriptional Silencing ◽  
S Phase ◽  
Nuclear Antigen ◽  
Histone Chaperones ◽  
Replication Forks ◽  
Nucleosome Assembly ◽  
Loading And Unloading ◽  
Proliferating Cell

In Saccharomyces cerevisiae, heterochromatin structures required for transcriptional silencing of the HML and HMR loci are duplicated in coordination with passing DNA replication forks. Despite major reorganization of chromatin structure, the heterochromatic, transcriptionally silent states of HML and HMR are successfully maintained throughout S-phase. Mutations of specific components of the replisome diminish the capacity to maintain silencing of HML and HMR through replication. Similarly, mutations in histone chaperones involved in replication-coupled nucleosome assembly reduce gene silencing. Bridging these observations, we determined that the proliferating cell nuclear antigen (PCNA) unloading activity of Elg1 was important for coordinating DNA replication forks with the process of replication-coupled nucleosome assembly to maintain silencing of HML and HMR through S-phase. Collectively, these data identified a mechanism by which chromatin reassembly is coordinated with DNA replication to maintain silencing through S-phase.

scholarly journals TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

2015 ◽  
Vol 212 (1) ◽  
pp. 63-75 ◽  
Author(s):  
Saskia Hoffmann ◽  
Stine Smedegaard ◽  
Kyosuke Nakamura ◽  
Gulnahar B. Mortuza ◽  
Markus Räschle ◽  
...  
Keyword(s):  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Genome Stability ◽  
Replication Stress ◽  
Nuclear Antigen ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Checkpoint Signaling ◽  
Processivity Factor ◽  
Proliferating Cell

Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication.

scholarly journals A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

1995 ◽  
Vol 15 (8) ◽  
pp. 4420-4429 ◽  
Author(s):  
R Ayyagari ◽  
K J Impellizzeri ◽  
B L Yoder ◽  
S L Gary ◽  
P M Burgers
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Nuclear Antigen ◽  
Replication Factor C ◽  
Dna Polymerase Delta ◽  
Cold Sensitive ◽  
Factor C ◽  
Proliferating Cell

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.

scholarly journals In Vivo Analysis Reveals That the Interdomain Region of the Yeast Proliferating Cell Nuclear Antigen Is Important for DNA Replication and DNA Repair

Genetics ◽  
1996 ◽  
Vol 144 (2) ◽  
pp. 479-493 ◽  
Author(s):  
Neelam S Amin ◽  
Connie Holm
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Velocity Sedimentation ◽  
Nuclear Antigen ◽  
Restrictive Temperature ◽  
Chromosomal Dna ◽  
Dna Breaks ◽  
Proliferating Cell

Abstract To identify the regions of the proliferating cell nuclear antigen (PCNA) that are important for function in vivo, we used random mutagenesis to isolate 10 cold-sensitive (Cs−) and 31 methyl methanesulfonate-sensitive (Mmss) mutations of the PCNA gene (POL30) in Saccharomyces cerevisiae. Unlike the Mmss mutations, the CsC mutations are strikingly clustered in the interdomain region of the three-dimensional PCNA monomer structure. At the restrictive temperature, the Cs−  pol30 mutants undergo a RAD9 dependent arrest as large-budded cells with a 2c DNA content. Defects in DNA synthesis are suggested by a significant delay in the progression of synchronized pol30 cells through S phase at the restrictive temperature. DNA repair defects are revealed by the observation that Cs−  pol30 mutants are very sensitive to the alkylating agent MMS and mildly sensitive to ultraviolet radiation, although they are not sensitive to gamma radiation. Finally, analysis of the chromosomal DNA in pol30 cells by velocity sedimentation gradients shows that pol30 cells accumulate single-stranded DNA breaks at the restrictive temperature. Thus, our results show that PCNA plays an essential role in both DNA replication and DNA repair in vivo.

scholarly journals Maneuvers on PCNA Rings during DNA Replication and Repair

Genes ◽  
2018 ◽  
Vol 9 (8) ◽  
pp. 416 ◽  
Author(s):  
Dea Slade
Keyword(s):  
Dna Replication ◽  
Protein Interactions ◽  
Proliferating Cell Nuclear Antigen ◽  
Binding Proteins ◽  
Nuclear Antigen ◽  
Vast Number ◽  
Post Translational Modifications ◽  
Dna Replication And Repair ◽  
Replicative Polymerases ◽  
Proliferating Cell

DNA replication and repair are essential cellular processes that ensure genome duplication and safeguard the genome from deleterious mutations. Both processes utilize an abundance of enzymatic functions that need to be tightly regulated to ensure dynamic exchange of DNA replication and repair factors. Proliferating cell nuclear antigen (PCNA) is the major coordinator of faithful and processive replication and DNA repair at replication forks. Post-translational modifications of PCNA, ubiquitination and acetylation in particular, regulate the dynamics of PCNA-protein interactions. Proliferating cell nuclear antigen (PCNA) monoubiquitination elicits ‘polymerase switching’, whereby stalled replicative polymerase is replaced with a specialized polymerase, while PCNA acetylation may reduce the processivity of replicative polymerases to promote homologous recombination-dependent repair. While regulatory functions of PCNA ubiquitination and acetylation have been well established, the regulation of PCNA-binding proteins remains underexplored. Considering the vast number of PCNA-binding proteins, many of which have similar PCNA binding affinities, the question arises as to the regulation of the strength and sequence of their binding to PCNA. Here I provide an overview of post-translational modifications on both PCNA and PCNA-interacting proteins and discuss their relevance for the regulation of the dynamic processes of DNA replication and repair.

scholarly journals Interactions between Telomerase and Primase Physically Link the Telomere and Chromosome Replication Machinery

2002 ◽  
Vol 22 (16) ◽  
pp. 5859-5868 ◽  
Author(s):  
Saugata Ray ◽  
Zemfira Karamysheva ◽  
Libin Wang ◽  
Dorothy E. Shippen ◽  
Carolyn M. Price
Keyword(s):  
De Novo ◽  
Telomerase Activity ◽  
Nuclear Antigen ◽  
Replication Machinery ◽  
Developmentally Regulated ◽  
Coordinate Regulation ◽  
Euplotes Crassus ◽  
Order Complexes ◽  
Proliferating Cell ◽  
Lagging Strand

ABSTRACT In the ciliate Euplotes crassus, millions of new telomeres are synthesized by telomerase and polymerase α-primase during macronuclear development in mated cells. Concomitant with de novo telomere formation, telomerase assembles into higher-order complexes of 550 kDa, 1,600 kDa, and 5 MDa. We show here that telomerase is physically associated with the lagging-strand replication machinery in these complexes. Antibodies against DNA primase precipitated telomerase activity from all three complexes from mated cells but not the 280-kDa telomerase complex from vegetatively growing cells. Moreover, when telomerase was affinity purified, primase copurified with enzyme from mated cells but not with the 280-kDa vegetative complex. Thus, the association of telomerase and primase is developmentally regulated. Intriguingly, PCNA (proliferating cell nuclear antigen) was also found in the 5-MDa complex from mated cells. We therefore speculate that this complex is a complete telomere synthesis machine, while the smaller complexes are assembly intermediates. The physical association of telomerase and primase explains the coordinate regulation of telomeric G- and C-strand synthesis and the efficiency of telomere addition in E. crassus.

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