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

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.

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The Tip of an Iceberg: Replication-Associated Functions of the Tumor Suppressor p53

Cancers ◽

10.3390/cancers10080250 ◽

2018 ◽

Vol 10 (8) ◽

pp. 250 ◽

Cited By ~ 7

Author(s):

Vanesa Gottifredi ◽

Lisa Wiesmüller

Keyword(s):

Dna Replication ◽

Tumor Suppressor ◽

Target Genes ◽

Data Sets ◽

Replication Forks ◽

Tumor Suppressor P53 ◽

Direct Role ◽

Associated Functions ◽

The Impact

The tumor suppressor p53 is a transcriptional factor broadly mutated in cancer. Most inactivating and gain of function mutations disrupt the sequence-specific DNA binding domain, which activates target genes. This is perhaps the main reason why most research has focused on the relevance of such transcriptional activity for the prevention or elimination of cancer cells. Notwithstanding, transcriptional regulation may not be the only mechanism underlying its role in tumor suppression and therapeutic responses. In the past, a direct role of p53 in DNA repair transactions that include the regulation of homologous recombination has been suggested. More recently, the localization of p53 at replication forks has been demonstrated and the effect of p53 on nascent DNA elongation has been explored. While some data sets indicate that the regulation of ongoing replication forks by p53 may be mediated by p53 targets such as MDM2 (murine double minute 2) and polymerase (POL) eta other evidences demonstrate that p53 is capable of controlling DNA replication by directly interacting with the replisome and altering its composition. In addition to discussing such findings, this review will also analyze the impact that p53-mediated control of ongoing DNA replication has on treatment responses and tumor suppressor abilities of this important anti-oncogene.

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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.

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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.

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Error-Free DNA Damage Tolerance and Sister Chromatid Proximity during DNA Replication Rely on the Polα/Primase/Ctf4 Complex

Molecular Cell ◽

10.1016/j.molcel.2014.12.038 ◽

2015 ◽

Vol 57 (5) ◽

pp. 812-823 ◽

Cited By ~ 80

Author(s):

Marco Fumasoni ◽

Katharina Zwicky ◽

Fabio Vanoli ◽

Massimo Lopes ◽

Dana Branzei

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Sister Chromatid ◽

Damage Tolerance ◽

Dna Damage Tolerance ◽

Free Dna

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MELK-T1, a small-molecule inhibitor of protein kinase MELK, decreases DNA-damage tolerance in proliferating cancer cells

Bioscience Reports ◽

10.1042/bsr20150194 ◽

2015 ◽

Vol 35 (6) ◽

Cited By ~ 39

Author(s):

Lijs Beke ◽

Cenk Kig ◽

Joannes T. M. Linders ◽

Shannah Boens ◽

An Boeckx ◽

...

Keyword(s):

Dna Damage ◽

Protein Kinase ◽

Cancer Cells ◽

Small Molecule ◽

Damage Tolerance ◽

Replication Stress ◽

Small Molecule Inhibitor ◽

Dna Damage Tolerance ◽

Molecule Inhibitor ◽

Senescence Phenotype

Protein kinase MELK has oncogenic properties and is highly overexpressed in some tumors. In the present study, we show that a novel MELK inhibitor causes both the inhibition and degradation of MELK, culminating in replication stress and a senescence phenotype.

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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.

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Non-Recombinogenic Functions of Rad51, BRCA2, and Rad52 in DNA Damage Tolerance

Genes ◽

10.3390/genes12101550 ◽

2021 ◽

Vol 12 (10) ◽

pp. 1550

Author(s):

Félix Prado

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Damage Tolerance ◽

Molecular Switches ◽

Strand Exchange ◽

Template Switching ◽

Replication Forks ◽

Lesion Bypass ◽

Dna Damage Tolerance ◽

Exchange Activity

The DNA damage tolerance (DDT) response is aimed to timely and safely complete DNA replication by facilitating the advance of replication forks through blocking lesions. This process is associated with an accumulation of single-strand DNA (ssDNA), both at the fork and behind the fork. Lesion bypass and ssDNA filling can be performed by translation synthesis (TLS) and template switching mechanisms. TLS uses low-fidelity polymerases to incorporate a dNTP opposite the blocking lesion, whereas template switching uses a Rad51/ssDNA nucleofilament and the sister chromatid to bypass the lesion. Rad51 is loaded at this nucleofilament by two mediator proteins, BRCA2 and Rad52, and these three factors are critical for homologous recombination (HR). Here, we review recent advances showing that Rad51, BRCA2, and Rad52 perform some of these functions through mechanisms that do not require the strand exchange activity of Rad51: the formation and protection of reversed fork structures aimed to bypass blocking lesions, and the promotion of TLS. These findings point to the central HR proteins as potential molecular switches in the choice of the mechanism of DDT.

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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.

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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.

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The activation loop residue serine 173 of S.pombe Chk1 kinase is critical for the response to DNA replication stress

10.1101/164244 ◽

2017 ◽

Author(s):

Naomi Coulton ◽

Thomas Caspari

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Stress ◽

Genetic Alterations ◽

Activation Loop ◽

Replication Forks ◽

Dna Polymerase Delta ◽

Dna Replication Stress ◽

Summary Statement ◽

Chk1 Kinase

AbstractWhy the DNA damage checkpoint kinase Chk1 protects the genome of lower and higher eukaryotic cells differentially is still unclear. Mammalian Chk1 regulates replication origins, safeguards DNA replication forks and promotes fork progression. Conversely, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A) in the activation loop of fission yeast Chk1 abolishes the G1-M and S-M checkpoints without affecting the G2-M arrest. Although Chk1-S173A is fully phosphorylated at serine 345 by the DNA damage sensor Rad3 (ATR) when DNA replication forks break, cells fail to stop the cell cycle. Mutant cells are uniquely sensitive to the DNA alkylation agent methyl- methanesulfate (MMS). This MMS sensitivity is genetically linked with the lagging strand DNA polymerase delta. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 is impaired. Serine 173 is equivalent to lysine 166 in human Chk1, an amino acid important for substrate specificity. We conclude that the removal of serine 173 impairs the phosphorylation of a Chk1 target that is important to protect cells from DNA replication stress.Summary statementMutation of serine-173 in the activation loop of Chk1 kinase may promote cancer as it abolishes the response to genetic alterations that arise while chromosomes are being copied.

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