PIDD orchestrates translesion DNA synthesis in response to UV irradiation.
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ABSTRACT:
SUBMITTER: Logette E
PROVIDER: S-EPMC3131944 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Deficiency of Parkin is a major cause of early-onset Parkinson's disease (PD). Notably, PD patients also exhibit a significantly higher risk in melanoma and other skin tumors, while the mechanism remains largely unknown. In this study, we show that depletion of Parkin causes compromised cell viability and genome stability after ultraviolet (UV) radiation. We demonstrate that Parkin promotes efficient Rad18-dependent proliferating cell nuclear antigen (PCNA) monoubiquitination by facilitating the formation of Replication protein A (RPA)-coated ssDNA upon UV radiation. Furthermore, Parkin is found to physically interact with NBS1 (Nijmegen breakage syndrome 1), and to be required for optimal recruitment of NBS1 and DNA polymerase eta (Polη) to UV-induced damage sites. Consequently, depletion of Parkin leads to increased UV-induced mutagenesis. These findings unveil an important role of Parkin in protecting genome stability through positively regulating translesion DNA synthesis (TLS) upon UV damage, providing a novel mechanistic link between Parkin deficiency and predisposition to skin cancers in PD patients.
| S-EPMC5482665 | biostudies-literature
Project description:In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa-Atr-Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects.
| S-EPMC4395481 | biostudies-literature
Project description:All living organisms are continually exposed to agents that damage their DNA, which threatens the integrity of their genome. As a consequence, cells are equipped with a plethora of DNA repair enzymes to remove the damaged DNA. Unfortunately, situations nevertheless arise where lesions persist, and these lesions block the progression of the cell's replicase. In these situations, cells are forced to choose between recombination-mediated "damage avoidance" pathways or a specialized DNA polymerase (pol) to traverse the blocking lesion. The latter process is referred to as Translesion DNA Synthesis (TLS). As inferred by its name, TLS not only results in bases being (mis)incorporated opposite DNA lesions but also bases being (mis)incorporated downstream of the replicase-blocking lesion, so as to ensure continued genome duplication and cell survival. Escherichia coli and Salmonella typhimurium possess five DNA polymerases, and while all have been shown to facilitate TLS under certain experimental conditions, it is clear that the LexA-regulated and damage-inducible pols II, IV, and V perform the vast majority of TLS under physiological conditions. Pol V can traverse a wide range of DNA lesions and performs the bulk of mutagenic TLS, whereas pol II and pol IV appear to be more specialized TLS polymerases.
| S-EPMC4231548 | biostudies-literature
Project description:Ultraviolet-induced 6-4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD) can be tolerated by translesion DNA polymerases (TLS Pols) at stalled replication forks or by gap-filling. Here, we investigated the involvement of Pol?, Rev1 and Rev3L (Pol? catalytic subunit) in the specific bypass of 6-4PP and CPD in repair-deficient XP-C human cells. We combined DNA fiber assay and novel methodologies for detection and quantification of single-stranded DNA (ssDNA) gaps on ongoing replication forks and postreplication repair (PRR) tracts in the human genome. We demonstrated that Rev3L, but not Rev1, is required for postreplicative gap-filling, while Pol? and Rev1 are responsible for TLS at stalled replication forks. Moreover, specific photolyases were employed to show that in XP-C cells, CPD arrest replication forks, while 6-4PP are responsible for the generation of ssDNA gaps and PRR tracts. On the other hand, in the absence of Pol? or Rev1, both types of lesion block replication forks progression. Altogether, the data directly show that, in the human genome, Pol? and Rev1 bypass CPD and 6-4PP at replication forks, while only 6-4PP are also tolerated by a Pol?-dependent gap-filling mechanism, independent of S phase.
| S-EPMC4937316 | biostudies-literature
Project description:Translesion DNA synthesis (TLS) can use specialized DNA polymerases to insert and/or extend nucleotides across lesions, thereby limiting stalled replication fork collapse and the potential for cell death. Recent studies have shown that monoubiquitinated proliferating cell nuclear antigen (PCNA) plays an important role in recruitment of Y-family TLS polymerases to stalled replication forks after DNA damage treatment. To explore the possible roles of other factors that regulate the ultraviolet (UV)-induced assembly of specialized DNA polymerases at arrested replication forks, we performed immunoprecipitation experiments combined with mass spectrometry and established that DNA polymerase kappa (Polκ) can partner with MSH2, an important mismatch repair protein associated with hereditary non-polyposis colorectal cancer. We found that depletion of MSH2 impairs PCNA monoubiquitination and the formation of foci containing Polκ and other TLS polymerases after UV irradiation of cells. Interestingly, expression of MSH2 in Rad18-deficient cells increased UV-induced Polκ and REV1 focus formation without detectable changes in PCNA monoubiquitination, indicating that MSH2 can regulate post-UV focus formation by specialized DNA polymerases in both PCNA monoubiquitination-dependent and -independent fashions. Moreover, we observed that MSH2 can facilitate TLS across cyclobutane pyrimidine dimers photoproducts in living cells, presenting a novel role of MSH2 in post-UV cellular responses.
| S-EPMC3905884 | biostudies-literature
Project description:In response to DNA damage such as from UV irradiation, mammalian Y-family translesion synthesis (TLS) polymerases Pol? and Rev1 colocalize with proliferating cell nuclear antigen at nuclear foci, presumably representing stalled replication sites. However, it is unclear whether the localization of one polymerase is dependent on another. Furthermore, there is no report on the in vivo characterization of the Rev3 catalytic subunit of the B-family TLS polymerase Pol?. Here we describe the detection of endogenous human Pol?, Rev1, and Rev3 by immunocytochemistry using existing or newly created antibodies, as well as various means of inhibiting their expression, which allows us to examine the dynamics of endogenous TLS polymerases in response to UV irradiation. It is found that Rev1 and Pol? are independently recruited to the nuclear foci, whereas the Rev3 nuclear focus formation requires Rev1 but not Pol?. In contrast, neither Rev1 nor Pol? recruitment requires Rev3. To further support these conclusions, we find that simultaneous suppression of Pol? and Rev3 results in an additive cellular sensitivity to UV irradiation. These observations suggest a cooperative and sequential assembly of TLS polymerases in response to DNA damage. They also support and extend the current polymerase switch model.
| S-EPMC3128538 | biostudies-literature
Project description:Exposure to ultraviolet light induces a number of forms of damage in DNA, of which (6-4) photoproducts present the most formidable challenge to DNA replication. No single DNA polymerase has been shown to bypass these lesions efficiently in vitro suggesting that the coordinate use of a number of different enzymes is required in vivo. To further understand the mechanisms and control of lesion bypass in vivo, we have devised a plasmid-based system to study the replication of site-specific T-T(6-4) photoproducts in chicken DT40 cells. We show that DNA polymerase zeta is absolutely required for translesion synthesis (TLS) of this lesion, while loss of DNA polymerase eta has no detectable effect. We also show that either the polymerase-binding domain of REV1 or ubiquitinated PCNA is required for the recruitment of Polzeta as the catalytic TLS polymerase. Finally, we demonstrate a previously unappreciated role for REV1 in ensuring bypass synthesis remains in frame with the template. Our data therefore suggest that REV1 not only helps to coordinate the delivery of DNA polymerase zeta to a stalled primer terminus but also restrains its activity to ensure that nucleotides are incorporated in register with the template strand.
| S-EPMC2588525 | biostudies-literature
Project description:Although the primary function of DNA polymerase (pol) β is associated with gap-filling DNA synthesis as part of the DNA base excision repair pathway, translesion synthesis activity has also been described. To further understand the potential role of pol β-catalyzed translesion DNA synthesis (TLS) and the structure-function relationships of specific residues in pol β, wild-type and selected mutants of pol β were used in TLS assays with DNA substrates containing bulky polycyclic aromatic hydrocarbon-adducted oligonucleotides. Stereospecific (+) and (-)-anti-trans-(C(10)S and C(10)R) benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (BPDE) adducts were covalently attached to both the N(6)-adenine and N(2)-guanine in the major and minor grooves, respectively. For all substrates tested, the presence of the BPDE adducts greatly decreased the efficiency of nucleotide incorporation opposite the lesion, and the stereochemistry of the adducts also further modulated the efficiency of the insertion step, such that lesions which were oriented in the 3' direction relative to the approaching polymerase were considerably more blocking than those oriented in the 5' direction. In the absence of a downstream DNA strand, the extension step beyond the adduct was extremely inefficient, relative to a dinucleotide gap-filling reaction, such that in the presence of the downstream DNA, dinucleotide incorporation was strongly favored. In general, analyses of the TLS activities of four pol β mutants revealed similar overall properties, but wild-type pol β exhibited more than 50-fold greater extension and bypass of the C(10)S-dA adducts as compared to a low fidelity mutant R283K expected to interact with the templating base. Replication bypass investigations were further extended to include analyses of HIV-1 reverse transcriptase, and these studies revealed patterns of inhibition very similar to that observed for pol β.
| S-EPMC3523550 | biostudies-other
Project description:Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. It remains unclear if the role of DDR is solely in meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7-/-) result in a >150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both DNA demethylation, a critical step in germline epigenetic reprogramming, and in activation of the germ cell transcriptional program. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for completion of DNA demethylation and the progression of PGC development.
2024-04-15 | GSE253991 | GEO
Project description:The well-being of all living organisms relies on the accurate duplication of their genomes. This is usually achieved by highly elaborate replicase complexes which ensure that this task is accomplished timely and efficiently. However, cells often must resort to the help of various additional "specialized" DNA polymerases that gain access to genomic DNA when replication fork progression is hindered. One such specialized polymerase family consists of the so-called "translesion synthesis" (TLS) polymerases; enzymes that have evolved to replicate damaged DNA. To fulfill their main cellular mission, TLS polymerases often must sacrifice precision when selecting nucleotide substrates. Low base-substitution fidelity is a well-documented inherent property of these enzymes. However, incorrect nucleotide substrates are not only those which do not comply with Watson-Crick base complementarity, but also those whose sugar moiety is incorrect. Does relaxed base-selectivity automatically mean that the TLS polymerases are unable to efficiently discriminate between ribonucleoside triphosphates and deoxyribonucleoside triphosphates that differ by only a single atom? Which strategies do TLS polymerases employ to select suitable nucleotide substrates? In this review, we will collate and summarize data accumulated over the past decade from biochemical and structural studies, which aim to answer these questions.
| S-EPMC6261451 | biostudies-literature