scholarly journals Functional and Physical Interaction of Yeast Mgs1 with PCNA: Impact on RAD6-Dependent DNA Damage Tolerance

2006 ◽  
Vol 26 (14) ◽  
pp. 5509-5517 ◽  
Author(s):  
Takashi Hishida ◽  
Tomoko Ohya ◽  
Yoshino Kubota ◽  
Yusuke Kamada ◽  
Hideo Shinagawa
Keyword(s):  
Dna Damage ◽  
Posttranslational Modifications ◽  
Cell Cycle Progression ◽  
Damage Tolerance ◽  
Genome Instability ◽  
Dna Helicase ◽  
Nuclear Antigen ◽  
Physical Interaction ◽  
Dna Damage Tolerance ◽  
Exogenous Dna

ABSTRACT Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication, DNA repair, cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1, Srs2, and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.

scholarly journals Helicase-Like Transcription Factor HLTF and E3 Ubiquitin Ligase SHPRH Confer DNA Damage Tolerance through Direct Interactions with Proliferating Cell Nuclear Antigen (PCNA)

2020 ◽  
Vol 21 (3) ◽  
pp. 693 ◽  
Author(s):  
Mareike Seelinger ◽  
Marit Otterlei
Keyword(s):  
Dna Damage ◽  
Transcription Factor ◽  
Damage Tolerance ◽  
Genome Instability ◽  
Ring Finger ◽  
Nuclear Antigen ◽  
Common Mutation ◽  
Dna Damage Tolerance ◽  
Replicative Stress ◽  
Template Switch

To prevent replication fork collapse and genome instability under replicative stress, DNA damage tolerance (DDT) mechanisms have evolved. The RAD5 homologs, HLTF (helicase-like transcription factor) and SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase), both ubiquitin ligases, are involved in several DDT mechanisms; DNA translesion synthesis (TLS), fork reversal/remodeling and template switch (TS). Here we show that these two human RAD5 homologs contain functional APIM PCNA interacting motifs. Our results show that both the role of HLTF in TLS in HLTF overexpressing cells, and nuclear localization of SHPRH, are dependent on interaction of HLTF and SHPRH with PCNA. Additionally, we detected multiple changes in the mutation spectra when APIM in overexpressed HLTF or SHPRH were mutated compared to overexpressed wild type proteins. In plasmids from cells overexpressing the APIM mutant version of HLTF, we observed a decrease in C to T transitions, the most common mutation caused by UV irradiation, and an increase in mutations on the transcribed strand. These results strongly suggest that direct binding of HLTF and SHPRH to PCNA is vital for their function in DDT.

DNA-damage tolerance through PCNA ubiquitination and sumoylation

2020 ◽  
Vol 477 (14) ◽  
pp. 2655-2677
Author(s):  
Li Fan ◽  
Tonghui Bi ◽  
Linxiao Wang ◽  
Wei Xiao
Keyword(s):  
Dna Damage ◽  
Posttranslational Modifications ◽  
Damage Tolerance ◽  
Nuclear Antigen ◽  
Replication Forks ◽  
Dna Damage Tolerance ◽  
Yeast Saccharomyces Cerevisiae ◽  
Dna Damaging Agents ◽  
Template Switch ◽  
Proliferating Cell

DNA-damage tolerance (DDT) is employed by eukaryotic cells to bypass replication-blocking lesions induced by DNA-damaging agents. In budding yeast Saccharomyces cerevisiae, DDT is mediated by RAD6 epistatic group genes and the central event for DDT is sequential ubiquitination of proliferating cell nuclear antigen (PCNA), a DNA clamp required for replication and DNA repair. DDT consists of two parallel pathways: error-prone DDT is mediated by PCNA monoubiquitination, which recruits translesion synthesis DNA polymerases to bypass lesions with decreased fidelity; and error-free DDT is mediated by K63-linked polyubiquitination of PCNA at the same residue of monoubiquitination, which facilitates homologous recombination-mediated template switch. Interestingly, the same PCNA residue is also subjected to sumoylation, which leads to inhibition of unwanted recombination at replication forks. All three types of PCNA posttranslational modifications require dedicated conjugating and ligation enzymes, and these enzymes are highly conserved in eukaryotes, from yeast to human.

scholarly journals Maturation of telomere 3’-overhangs is linked to the replication stress response

2020 ◽  
Author(s):  
Erin E. Henninger ◽  
Pascale Jolivet ◽  
Emilie Fallet ◽  
Mohcen Benmounah ◽  
Zhou Xu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Stress Response ◽  
Ubiquitin Ligase ◽  
Replication Fork ◽  
Damage Tolerance ◽  
Dna Helicase ◽  
Dna Damage Tolerance ◽  
Telomeric Repeats ◽  
Telomere Replication

AbstractPassage of the replication fork through telomeric repeats necessitates additional DNA processing by DNA repair factors, to regenerate the terminal 3’-overhang structure at leading telomeres. These factors are prevented from promoting telomeric recombination or fusion by an uncharacterized mechanism. Here we show that Rad5, a DNA helicase and ubiquitin ligase involved in the DNA damage tolerance pathway, participates in this mechanism. Rad5 is enriched at telomeres during telomere replication. Accelerated senescence seen in the absence of telomerase and Rad5, can be compensated for by a pathway involving the Rad51 recombinase and counteracted by the helicase Srs2. However, this pathway is only active at short telomeres. Instead, the ubiquitous activity of Rad5 during telomere replication is necessary for the proper reconstitution of the telomeric 3’-overhang, indicating that Rad5 is required to coordinate telomere maturation during telomere replication.

scholarly journals Mechanisms of DNA Damage Tolerance: Post-Translational Regulation of PCNA

Genes ◽  
2018 ◽  
Vol 10 (1) ◽  
pp. 10 ◽  
Author(s):  
Wendy Leung ◽  
Ryan Baxley ◽  
George-Lucian Moldovan ◽  
Anja-Katrin Bielinsky
Keyword(s):  
Dna Damage ◽  
Translational Regulation ◽  
Damage Tolerance ◽  
Somatic Hypermutation ◽  
Critical Role ◽  
Nuclear Antigen ◽  
Template Switching ◽  
Dna Damage Tolerance ◽  
Class Switch ◽  
Dna Lesion

DNA damage is a constant source of stress challenging genomic integrity. To ensure faithful duplication of our genomes, mechanisms have evolved to deal with damage encountered during replication. One such mechanism is referred to as DNA damage tolerance (DDT). DDT allows for replication to continue in the presence of a DNA lesion by promoting damage bypass. Two major DDT pathways exist: error-prone translesion synthesis (TLS) and error-free template switching (TS). TLS recruits low-fidelity DNA polymerases to directly replicate across the damaged template, whereas TS uses the nascent sister chromatid as a template for bypass. Both pathways must be tightly controlled to prevent the accumulation of mutations that can occur from the dysregulation of DDT proteins. A key regulator of error-prone versus error-free DDT is the replication clamp, proliferating cell nuclear antigen (PCNA). Post-translational modifications (PTMs) of PCNA, mainly by ubiquitin and SUMO (small ubiquitin-like modifier), play a critical role in DDT. In this review, we will discuss the different types of PTMs of PCNA and how they regulate DDT in response to replication stress. We will also cover the roles of PCNA PTMs in lagging strand synthesis, meiotic recombination, as well as somatic hypermutation and class switch recombination.

scholarly journals Control of PCNA deubiquitylation in yeast

2010 ◽  
Vol 38 (1) ◽  
pp. 104-109 ◽  
Author(s):  
Alfonso Gallego-Sánchez ◽  
Francisco Conde ◽  
Pedro San Segundo ◽  
Avelino Bueno
Keyword(s):  
Dna Damage ◽  
Crucial Role ◽  
Proliferating Cell Nuclear Antigen ◽  
Molecular Mechanisms ◽  
Damage Tolerance ◽  
Nuclear Antigen ◽  
Dna Damage Tolerance ◽  
Sliding Clamp ◽  
Evolutionarily Conserved ◽  
Proliferating Cell

Eukaryotes ubiquitylate the replication factor PCNA (proliferating-cell nuclear antigen) so that it tolerates DNA damage. Although, in the last few years, the understanding of the evolutionarily conserved mechanism of ubiquitylation of PCNA, and its crucial role in DNA damage tolerance, has progressed impressively, little is known about the deubiquitylation of this sliding clamp in most organisms. In the present review, we will discuss potential molecular mechanisms regulating PCNA deubiquitylation in yeast.

scholarly journals Mdt1, a Novel Rad53 FHA1 Domain-Interacting Protein, Modulates DNA Damage Tolerance and G2/M Cell Cycle Progression in Saccharomycescerevisiae

2004 ◽  
Vol 24 (7) ◽  
pp. 2779-2788 ◽  
Author(s):  
Brietta L. Pike ◽  
Suganya Yongkiettrakul ◽  
Ming-Daw Tsai ◽  
Jörg Heierhorst
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Progression ◽  
Structural Integrity ◽  
Damage Tolerance ◽  
Rna Recognition Motif ◽  
M Cell ◽  
Dna Damage Tolerance ◽  
Cycle Progression ◽  
Dependent Cell

ABSTRACT The Rad53 kinase plays a central role in yeast DNA damage checkpoints. Rad53 contains two FHA phosphothreonine-binding domains that are required for Rad53 activation and possibly downstream signaling. Here we show that the N-terminal Rad53 FHA1 domain interacts with the RNA recognition motif, coiled-coil, and SQ/TQ cluster domain-containing protein Mdt1 (YBl051C). The interaction of Rad53 and Mdt1 depends on the structural integrity of the FHA1 phosphothreonine-binding site as well as threonine-305 of Mdt1. Mdt1 is constitutively threonine phosphorylated and hyperphosphorylated in response to DNA damage in vivo. DNA damage-dependent Mdt1 hyperphosphorylation depends on the Mec1 and Tel1 checkpoint kinases, and Mec1 can directly phosphorylate a recombinant Mdt1 SQ/TQ domain fragment. MDT1 overexpression is synthetically lethal with a rad53 deletion, whereas mdt1 deletion partially suppresses the DNA damage hypersensitivity of checkpoint-compromised strains and generally improves DNA damage tolerance. In the absence of DNA damage, mdt1 deletion leads to delayed anaphase completion, with an elongated cell morphology reminiscent of that of G2/M cell cycle mutants. mdt1-dependent and DNA damage-dependent cell cycle delays are not additive, suggesting that they act in the same pathway. The data indicate that Mdt1 is involved in normal G2/M cell cycle progression and is a novel target of checkpoint-dependent cell cycle arrest pathways.

scholarly journals Dissecting PCNA function with a systematically designed mutation library in yeast

2018 ◽  
Author(s):  
Qingwen Jiang ◽  
Weimin Zhang ◽  
Chenghao Liu ◽  
Yicong Lin ◽  
Qingyu Wu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Damage Tolerance ◽  
Nuclear Antigen ◽  
Regulatory Function ◽  
Dna Metabolism ◽  
Dna Damage Tolerance ◽  
Functional Sites ◽  
Relationship Analysis ◽  
Biochemical Analyses ◽  
Partner Proteins

AbstractProliferating cell nuclear antigen (PCNA), encoded by POL30 in Saccharomyces cerevisiae, is a key component of DNA metabolism. Here a library consisting of 308 PCNA mutants was designed and synthesized to probe the contribution of each residue to its biological function. Five regions were identified with elevated sensitivity to DNA damaging reagents using high-throughput phenotype screening. Using a series of genetic and biochemical analyses, we demonstrated that one particular mutant, K168A, which displayed severe DNA damage sensitivity, abolished the DNA damage tolerance (DDT) pathway by disrupting interactions between PCNA and Rad5p. Subsequent domain analysis showed that the PCNA/Rad5p interaction is prerequisite for the function of Rad5p in DDT. Our study not only provides a resource in the form of a library of versatile mutants to study PCNA functions, but also reveals a key regulatory function of Rad5p, which highlights the importance of the PCNA-Rad5p interaction.Author summaryPCNA is regarded as the maestro of DNA replication fork because of the astonishing ability to interact with lots of partner proteins that participate in various DNA metabolism processes. However, it has remained elusive as to how does PCNA orchestrate these functions in harmony. Here, we constructed a systematic mutation library of PCNA, which covers every amino acid to map the functional sites of it. This carefully designed synthetic mutant pool could be generally useful and serve as a flexible resource, such as dissecting the functional mechanism of PCNA by genetic relationship analysis with key proteins through Synthetic genetic array. We further dissected the intrinsic mechanism for damage sensitivity of PCNAK168A, the most severe DNA damage sensitive mutant in our alanine scanning mutation library, this helps us to get better understanding of how PCNA participates in DNA damage tolerance (DDT) pathways. Our findings indicate that K168 site is vital for the interaction between DDT related partner proteins and PCNA, and also highlight the importance of the PCNA-Rad5p interaction.

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