Dissecting PCNA function with a systematically designed mutation library in yeast

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.

Download Full-text

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

International Journal of Molecular Sciences ◽

10.3390/ijms21030693 ◽

2020 ◽

Vol 21 (3) ◽

pp. 693 ◽

Cited By ~ 8

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.00307-06 ◽

2006 ◽

Vol 26 (14) ◽

pp. 5509-5517 ◽

Cited By ~ 48

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.

Download Full-text

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

Genes ◽

10.3390/genes10010010 ◽

2018 ◽

Vol 10 (1) ◽

pp. 10 ◽

Cited By ~ 31

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.

Download Full-text

Control of PCNA deubiquitylation in yeast

Biochemical Society Transactions ◽

10.1042/bst0380104 ◽

2010 ◽

Vol 38 (1) ◽

pp. 104-109 ◽

Cited By ~ 7

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.

Download Full-text

TheSaccharomyces cerevisiae rev6-1Mutation, Which Inhibits Both the Lesion Bypass and the Recombination Mode of DNA Damage Tolerance, Is an Allele ofPOL30, Encoding Proliferating Cell Nuclear Antigen

Genetics ◽

10.1534/genetics.106.058545 ◽

2006 ◽

Vol 173 (4) ◽

pp. 1983-1989 ◽

Cited By ~ 23

Author(s):

Hengshan Zhang ◽

Peter E. M. Gibbs ◽

Christopher W. Lawrence

Keyword(s):

Dna Damage ◽

Proliferating Cell Nuclear Antigen ◽

Damage Tolerance ◽

Nuclear Antigen ◽

Lesion Bypass ◽

Dna Damage Tolerance ◽

Proliferating Cell

Download Full-text

DNA-damage tolerance through PCNA ubiquitination and sumoylation

Biochemical Journal ◽

10.1042/bcj20190579 ◽

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.

Download Full-text

The ADP-ribosyltransferase PARP10/ARTD10 Interacts with Proliferating Cell Nuclear Antigen (PCNA) and Is Required for DNA Damage Tolerance

Journal of Biological Chemistry ◽

10.1074/jbc.m114.556340 ◽

2014 ◽

Vol 289 (19) ◽

pp. 13627-13637 ◽

Cited By ~ 49

Author(s):

Claudia M. Nicolae ◽

Erin R. Aho ◽

Alexander H. S. Vlahos ◽

Katherine N. Choe ◽

Subhajyoti De ◽

...

Keyword(s):

Dna Damage ◽

Proliferating Cell Nuclear Antigen ◽

Damage Tolerance ◽

Nuclear Antigen ◽

Dna Damage Tolerance ◽

Adp Ribosyltransferase ◽

Proliferating Cell

Download Full-text

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1605828113 ◽

2016 ◽

Vol 113 (30) ◽

pp. E4311-E4319 ◽

Cited By ~ 44

Author(s):

Stephanie Hampp ◽

Tina Kiessling ◽

Kerstin Buechle ◽

Sabrina F. Mansilla ◽

Jürgen Thomale ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Tumor Suppressor ◽

Damage Tolerance ◽

Replication Stress ◽

Nuclear Antigen ◽

Replication Forks ◽

Tumor Suppressor P53 ◽

Dna Damage Tolerance ◽

Direct Role

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker–induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

Download Full-text