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

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.

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Mechanisms for Maintaining Eukaryotic Replisome Progression in the Presence of DNA Damage

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.712971 ◽

2021 ◽

Vol 8 ◽

Author(s):

Thomas A. Guilliam

Keyword(s):

Dna Damage ◽

Single Molecule ◽

Replication Fork ◽

Genome Stability ◽

Genome Duplication ◽

Damage Tolerance ◽

Tolerance Mechanisms ◽

Dna Damage Tolerance ◽

Replication Fork Progression ◽

The Impact

The eukaryotic replisome coordinates template unwinding and nascent-strand synthesis to drive DNA replication fork progression and complete efficient genome duplication. During its advancement along the parental template, each replisome may encounter an array of obstacles including damaged and structured DNA that impede its progression and threaten genome stability. A number of mechanisms exist to permit replisomes to overcome such obstacles, maintain their progression, and prevent fork collapse. A combination of recent advances in structural, biochemical, and single-molecule approaches have illuminated the architecture of the replisome during unperturbed replication, rationalised the impact of impediments to fork progression, and enhanced our understanding of DNA damage tolerance mechanisms and their regulation. This review focusses on these studies to provide an updated overview of the mechanisms that support replisomes to maintain their progression on an imperfect template.

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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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Replication intermediate architecture reveals the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells

10.1101/2020.10.12.336107 ◽

2020 ◽

Author(s):

Yann Benureau ◽

Caroline Pouvelle ◽

Eliana Moreira Tavares ◽

Pauline Dupaigne ◽

Emmanuelle Despras ◽

...

Keyword(s):

Dna Damage ◽

Dna Synthesis ◽

Replication Fork ◽

Damage Tolerance ◽

Dna Lesions ◽

Human Cells ◽

Compensatory Mechanism ◽

Dna Damage Tolerance ◽

Replication Intermediate

AbstractDNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remain elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.

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

Genes ◽

10.3390/genes9120614 ◽

2018 ◽

Vol 9 (12) ◽

pp. 614 ◽

Cited By ~ 1

Author(s):

Takuya Abe ◽

Dana Branzei ◽

Kouji Hirota

Keyword(s):

Dna Damage ◽

Dna Replication ◽

B Lymphocyte ◽

Damage Tolerance ◽

Base Pairs ◽

Dna Damage Tolerance ◽

Abasic Sites ◽

Activation Induced Deaminase ◽

Template Switch ◽

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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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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