DNA Damage Tolerance Mechanisms Revealed from the Analysis of Immunoglobulin V Gene Diversification in Avian DT40 Cells

DNA replication is an essential biochemical reaction in dividing cells that frequently stalls at damaged sites. Homologous/homeologous recombination (HR)-mediated template switch and translesion DNA synthesis (TLS)-mediated bypass processes release arrested DNA replication forks. These mechanisms are pivotal for replication fork maintenance and play critical roles in DNA damage tolerance (DDT) and gap-filling. The avian DT40 B lymphocyte cell line provides an opportunity to examine HR-mediated template switch and TLS triggered by abasic sites by sequencing the constitutively diversifying immunoglobulin light-chain variable gene (IgV). During IgV diversification, activation-induced deaminase (AID) converts dC to dU, which in turn is excised by uracil DNA glycosylase and yields abasic sites within a defined window of around 500 base pairs. These abasic sites can induce gene conversion with a set of homeologous upstream pseudogenes via the HR-mediated template switch, resulting in templated mutagenesis, or can be bypassed directly by TLS, resulting in non-templated somatic hypermutation at dC/dG base pairs. In this review, we discuss recent works unveiling IgV diversification mechanisms in avian DT40 cells, which shed light on DDT mode usage in vertebrate cells and tolerance of abasic sites.

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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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PCNA Ubiquitylation: Instructive or Permissive to DNA Damage Tolerance Pathways?

Biomolecules ◽

10.3390/biom11101543 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1543

Author(s):

Jun Che ◽

Xin Hong ◽

Hai Rao

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Damage Tolerance ◽

Translesion Synthesis ◽

Dna Lesions ◽

Template Switching ◽

Dna Damage Tolerance ◽

Future Studies ◽

Dna Lesion ◽

Replicative Polymerases

DNA lesions escaping from repair often block the DNA replicative polymerases required for DNA replication and are handled during the S/G2 phases by the DNA damage tolerance (DDT) mechanisms, which include the error-prone translesion synthesis (TLS) and the error-free template switching (TS) pathways. Where the mono-ubiquitylation of PCNA K164 is critical for TLS, the poly-ubiquitylation of the same residue is obligatory for TS. However, it is not known how cells divide the labor between TLS and TS. Due to the fact that the type of DNA lesion significantly influences the TLS and TS choice, we propose that, instead of altering the ratio between the mono- and poly-Ub forms of PCNA, the competition between TLS and TS would automatically determine the selection between the two pathways. Future studies, especially the single integrated lesion “i-Damage” system, would elucidate detailed mechanisms governing the choices of specific DDT pathways.

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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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Abstract 3722: Extracellular arginine starvation imposes DNA replication fork stall and permits DNA damage tolerance

10.1158/1538-7445.am2020-3722 ◽

2020 ◽

Author(s):

Yi-Chang Wang ◽

Andrew A. Kelso ◽

Yi-Hsuan Chen ◽

Chi-An Hsieh ◽

Wei-Kai Chen ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Fork ◽

Damage Tolerance ◽

Dna Damage Tolerance

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Access to PCNA by Srs2 and Elg1 Controls the Choice between Alternative Repair Pathways in Saccharomyces cerevisiae

mBio ◽

10.1128/mbio.00705-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 2

Author(s):

Matan Arbel ◽

Alex Bronstein ◽

Soumitra Sau ◽

Batia Liefshitz ◽

Martin Kupiec

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Damage Tolerance ◽

Dna Polymerases ◽

Strand Transfer ◽

Dna Damage Tolerance ◽

Dna Repair Mechanisms ◽

Dna Damaging Agents ◽

Recombination Protein ◽

Repair Mechanisms

ABSTRACT During DNA replication, stalling can occur when the replicative DNA polymerases encounter lesions or hard-to replicate regions. Under these circumstances, the processivity factor PCNA gets ubiquitylated at lysine 164, inducing the DNA damage tolerance (DDT) mechanisms that can bypass lesions encountered during DNA replication. PCNA can also be SUMOylated at the same residue or at lysine 127. Surprisingly, pol30-K164R mutants display a higher degree of sensitivity to DNA-damaging agents than pol30-KK127,164RR strains, unable to modify any of the lysines. Here, we show that in addition to translesion synthesis and strand-transfer DDT mechanisms, an alternative repair mechanism (“salvage recombination”) that copies information from the sister chromatid is repressed by the recruitment of Srs2 to SUMOylated PCNA. Overexpression of Elg1, the PCNA unloader, or of the recombination protein Rad52 allows its activation. We dissect the genetic requirements for this pathway, as well as the interactions between Srs2 and Elg1. IMPORTANCE PCNA, the ring that encircles DNA maintaining the processivity of DNA polymerases, is modified by ubiquitin and SUMO. Whereas ubiquitin is required for bypassing lesions through the DNA damage tolerance (DDT) pathways, we show here that SUMOylation represses another pathway, salvage recombination. The Srs2 helicase is recruited to SUMOylated PCNA and prevents the salvage pathway from acting. The pathway can be induced by overexpressing the PCNA unloader Elg1, or the homologous recombination protein Rad52. Our results underscore the role of PCNA modifications in controlling the various bypass and DNA repair mechanisms.

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Multiple biochemical properties of the p53 molecule contribute to activation of polymerase iota-dependent DNA damage tolerance

Nucleic Acids Research ◽

10.1093/nar/gkaa974 ◽

2020 ◽

Vol 48 (21) ◽

pp. 12188-12203

Author(s):

Stephanie Biber ◽

Helmut Pospiech ◽

Vanesa Gottifredi ◽

Lisa Wiesmüller

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Topoisomerase I ◽

Mutational Analysis ◽

Damage Tolerance ◽

Biochemical Properties ◽

Replication Proteins ◽

Dna Damage Tolerance ◽

C Terminus ◽

Cancer Tissues

Abstract We have previously reported that p53 decelerates nascent DNA elongation in complex with the translesion synthesis (TLS) polymerase ι (POLι) which triggers a homology-directed DNA damage tolerance (DDT) pathway to bypass obstacles during DNA replication. Here, we demonstrate that this DDT pathway relies on multiple p53 activities, which can be disrupted by TP53 mutations including those frequently found in cancer tissues. We show that the p53-mediated DDT pathway depends on its oligomerization domain (OD), while its regulatory C-terminus is not involved. Mutation of residues S315 and D48/D49, which abrogate p53 interactions with the DNA repair and replication proteins topoisomerase I and RPA, respectively, and residues L22/W23, which disrupt formation of p53-POLι complexes, all prevent this DDT pathway. Our results demonstrate that the p53-mediated DDT requires the formation of a DNA binding-proficient p53 tetramer, recruitment of such tetramer to RPA-coated forks and p53 complex formation with POLι. Importantly, our mutational analysis demonstrates that transcriptional transactivation is dispensable for the POLι-mediated DDT pathway, which we show protects against DNA replication damage from endogenous and exogenous sources.

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The Chromatin-binding Protein Spn1 contributes to Genome Instability in Saccharomyces cerevisiae

10.1101/341768 ◽

2018 ◽

Author(s):

Alison K. Thurston ◽

Catherine A. Radebaugh ◽

Adam R. Almeida ◽

Juan Lucas Argueso ◽

Laurie A. Stargell

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Dna Damage ◽

Genome Stability ◽

Damage Tolerance ◽

Genome Instability ◽

Chromatin Binding ◽

Dna Damage Tolerance ◽

Template Switch

AbstractCells expend a large amount of energy to maintain their DNA sequence. DNA repair pathways, cell cycle checkpoint activation, proofreading polymerases, and chromatin structure are ways in which the cell minimizes changes to the genome. During replication, the DNA damage tolerance pathway allows the replication forks to bypass damage on the template strand. This avoids prolonged replication fork stalling, which can contribute to genome instability. The DNA damage tolerance pathway includes two sub-pathways: translesion synthesis and template switch. Post-translational modification of PCNA and the histone tails, cell cycle phase, and local DNA structure have all been shown to influence sub-pathway choice. Chromatin architecture contributes to maintaining genome stability by providing physical protection of the DNA and by regulating DNA processing pathways. As such, chromatin-binding factors have been implicated in maintaining genome stability. Using Saccharomyces cerevisiae, we examined the role of Spn1, a chromatin binding and transcription elongation factor, in DNA damage tolerance. Expression of a mutant allele of SPN1 results in increased resistance to the DNA damaging agent methyl methanesulfonate, lower spontaneous and damage-induced mutation rates, along with increased chronological lifespan. We attribute these effects to an increased usage of the template switch branch of the DNA damage tolerance pathway in the spn1 strain. This provides evidence for a role of wild type Spn1 in promoting genome instability, as well as having ties to overcoming replication stress and contributing to chronological aging.

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p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

Cell Death and Disease ◽

10.1038/s41419-021-04224-3 ◽

2021 ◽

Vol 12 (10) ◽

Author(s):

Yitian Guo ◽

Melanie Rall-Scharpf ◽

Jean-Christophe Bourdon ◽

Lisa Wiesmüller ◽

Stephanie Biber

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Damage Tolerance ◽

Inhibitory Effect ◽

Dna Damage Tolerance ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Fork Stalling ◽

P53 Isoforms ◽

Truncated Isoforms

AbstractThe recently discovered p53-dependent DNA damage tolerance (DDT) pathway relies on its biochemical activities in DNA-binding, oligomerization, as well as complex formation with the translesion synthesis (TLS) polymerase iota (POLι). These p53-POLι complexes slow down nascent DNA synthesis for safe, homology-directed bypass of DNA replication barriers. In this study, we demonstrate that the alternative p53-isoforms p53β, p53γ, Δ40p53α, Δ133p53α, and Δ160p53α differentially affect this p53-POLι-dependent DDT pathway originally described for canonical p53α. We show that the C-terminal isoforms p53β and p53γ, comprising a truncated oligomerization domain (OD), bind PCNA. Conversely, N-terminally truncated isoforms have a reduced capacity to engage in this interaction. Regardless of the specific loss of biochemical activities required for this DDT pathway, all alternative isoforms were impaired in promoting POLι recruitment to PCNA in the chromatin and in decelerating DNA replication under conditions of enforced replication stress after Mitomycin C (MMC) treatment. Consistent with this, all alternative p53-isoforms no longer stimulated recombination, i.e., bypass of endogenous replication barriers. Different from the other isoforms, Δ133p53α and Δ160p53α caused a severe DNA replication problem, namely fork stalling even in untreated cells. Co-expression of each alternative p53-isoform together with p53α exacerbated the DDT pathway defects, unveiling impaired POLι recruitment and replication deceleration already under unperturbed conditions. Such an inhibitory effect on p53α was particularly pronounced in cells co-expressing Δ133p53α or Δ160p53α. Notably, this effect became evident after the expression of the isoforms in tumor cells, as well as after the knockdown of endogenous isoforms in human hematopoietic stem and progenitor cells. In summary, mimicking the situation found to be associated with many cancer types and stem cells, i.e., co-expression of alternative p53-isoforms with p53α, carved out interference with p53α functions in the p53-POLι-dependent DDT pathway.

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