Both genotoxic and non-genotoxic chemicals can act as carcinogens. However, while genotoxic compounds lead directly to mutations that promote unregulated cell growth, the mechanism by which ...non-genotoxic carcinogens lead to cellular transformation is poorly understood. Using a model non-genotoxic carcinogen, arsenic, we show here that exposure to arsenic inhibits mismatch repair (MMR) in human cells, possibly through its ability to stimulate epidermal growth factor receptor (EGFR)-dependent tyrosine phosphorylation of proliferating cellular nuclear antigen (PCNA). HeLa cells exposed to exogenous arsenic demonstrate a dose- and time-dependent increase in the levels of EGFR and tyrosine 211-phosphorylated PCNA. Cell extracts derived from arsenic-treated HeLa cells are defective in MMR, and unphosphorylated recombinant PCNA restores normal MMR activity to these extracts. These results suggest a model in which arsenic induces expression of EGFR, which in turn phosphorylates PCNA, and phosphorylated PCNA then inhibits MMR, leading to increased susceptibility to carcinogenesis. This study suggests a putative novel mechanism of action for arsenic and other non-genotoxic carcinogens.
Background: Exposure to arsenic is linked to increased risk of human cancer.
Results: Arsenic-treated cells have higher levels of EGFR and tyrosine-phosphorylated PCNA and reduced mismatch repair activity.
Conclusion: The carcinogenic effect of arsenic may be mediated by its ability to promote EGFR-dependent PCNA phosphorylation, thereby inhibiting mismatch repair.
Significance: Arsenic-stimulated posttranslational modification of PCNA represents a novel mechanism of action for non-genotoxic carcinogens.
Abstract
Cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS) is activated in cells with defective DNA damage repair and signaling (DDR) factors, but a direct role for DDR factors ...in regulating cGAS activation in response to micronuclear DNA is still poorly understood. Here, we provide novel evidence that Nijmegen breakage syndrome 1 (NBS1) protein, a well-studied DNA double-strand break (DSB) sensor—in coordination with Ataxia Telangiectasia Mutated (ATM), a protein kinase, and Carboxy-terminal binding protein 1 interacting protein (CtIP), a DNA end resection factor—functions as an upstream regulator that prevents cGAS from binding micronuclear DNA. When NBS1 binds to micronuclear DNA via its fork-head–associated domain, it recruits CtIP and ATM via its N- and C-terminal domains, respectively. Subsequently, ATM stabilizes NBS1’s interaction with micronuclear DNA, and CtIP converts DSB ends into single-strand DNA ends; these two key events prevent cGAS from binding micronuclear DNA. Additionally, by using a cGAS tripartite system, we show that cells lacking NBS1 not only recruit cGAS to a major fraction of micronuclear DNA but also activate cGAS in response to these micronuclear DNA. Collectively, our results underscore how NBS1 and its binding partners prevent cGAS from binding micronuclear DNA, in addition to their classical functions in DDR signaling.
Graphical Abstract
Graphical Abstract
Following micronuclei generation in response to genotoxic stress, NBS1 is recruited to micronuclear DNA and that promotes CtIP-mediated end resection, resulting in the inhibition of cGAS binding to the micronuclear DNA.
RNA nanotechnology is rapidly emerging. Due to advantageous pharmacokinetics and favorable in vivo biodistribution, RNA nanoparticles have shown promise in targeted delivery of therapeutics. RNA ...nanotechnology applies bottom-up assembly, thus elucidation of the mechanism of interaction between multiple components is of fundamental importance. The tendency of diminishing concern about RNA instability has accelerated by the finding of the novel thermostable three-way junction (3WJ) motif of the phi29 DNA-packaging motor. The kinetics of these three components, each averaging 18 nucleotides (nt), was investigated to elucidate the mechanism for producing the stable 3WJ. The three fragments coassembled into the 3WJ with extraordinary speed and affinity via a two-step reaction mechanism, 3WJ
+ 3WJ
↔ 3WJ
+ 3WJ
↔ 3WJ
The first step of reaction between 3WJ
and 3WJ
is highly dynamic since these two fragments only contain 8 nt for complementation. In the second step, the 3WJ
, which contains 17 nt complementary to the 3WJ
complex, locks the unstable 3WJ
complex into a highly stable 3WJ. The resulting pRNA-3WJ is more stable than any of the dimer species as shown in the much more rapid association rates and slowest dissociation rate constant. The second step occurs at a very high association rate that is difficult to quantify, resulting in a rapid formation of a stable 3WJ. Elucidation of the mechanism of three-component collision in producing the ultrastable 3WJ proves a promising platform for bottom-up assembly of RNA nanoparticles as a new class of anion polymers for material science, electronic elements, or therapeutic reagents.
•Histone mark H3K36me3 is required for mismatch repair in vivo.•PCNA phosphorylation promotes genome instability by inhibiting mismatch repair.•MSH2 deacetylation/ubiquitination by HDAC6 ...downregulates mismatch repair activity.
DNA mismatch repair (MMR) protects genome integrity by correcting DNA replication-associated mispairs, modulating DNA damage-induced cell cycle checkpoints and regulating homeologous recombination. Loss of MMR function leads to cancer development. This review describes progress in understanding how MMR is carried out in the context of chromatin and how chromatin organization/compaction, epigenetic mechanisms and posttranslational modifications of MMR proteins influence and regulate MMR in eukaryotic cells.
Trinucleotide repeat (TNR) expansions cause nearly 20 severe human neurological diseases which are currently untreatable. For some of these diseases, ongoing somatic expansions accelerate disease ...progression and may influence age of onset. This new knowledge emphasizes the importance of understanding the protein factors that drive expansions. Recent genetic evidence indicates that the mismatch repair factor MutSβ (Msh2-Msh3 complex) and the histone deacetylase HDAC3 function in the same pathway to drive triplet repeat expansions. Here we tested the hypothesis that HDAC3 deacetylates MutSβ and thereby activates it to drive expansions. The HDAC3-selective inhibitor RGFP966 was used to examine its biological and biochemical consequences in human tissue culture cells. HDAC3 inhibition efficiently suppresses repeat expansion without impeding canonical mismatch repair activity. Five key lysine residues in Msh3 are direct targets of HDAC3 deacetylation. In cells expressing Msh3 in which these lysine residues are mutated to arginine, the inhibitory effect of RGFP966 on expansions is largely bypassed, consistent with the direct deacetylation hypothesis. RGFP966 treatment does not alter MutSβ subunit abundance or complex formation but does partially control its subcellular localization. Deacetylation sites in Msh3 overlap a nuclear localization signal, and we show that localization of MutSβ is partially dependent on HDAC3 activity. Together, these results indicate that MutSβ is a key target of HDAC3 deacetylation and provide insights into an innovative regulatory mechanism for triplet repeat expansions. The results suggest expansion activity may be druggable and support HDAC3-selective inhibition as an attractive therapy in some triplet repeat expansion diseases.
MicroRNAs (miRNAs) are critical post-transcriptional regulators and are derived from hairpin-shaped primary transcripts via a series of processing steps. However, how the production of individual ...miRNAs is regulated remains largely unknown. Similarly, loss or overexpression of the key mismatch repair protein MutLα (MLH1-PMS2 heterodimer) leads to genome instability and tumorigenesis, but the mechanisms controlling MutLα expression are unknown. Here we demonstrate in vitro and in vivo that MLH1 and miR-422a participate in a feedback loop that regulates the level of both molecules. Using a defined in-vitro miRNA processing system, we show that MutLα stimulates the conversion of pri-miR-422a to pre-miR-422a, as well as the processing of other miRNAs tested, implicating MutLα as a general stimulating factor for miRNA biogenesis. This newly identified MutLα function requires its ATPase and pri-miRNA binding activities. In contrast, miR-422a downregulates MutLα levels by suppressing MLH1 expression through base pairing with the MLH1 3'-untranslated region. A model depicting this feedback mechanism is discussed.
Abstract only
Proliferating cell nuclear antigen (PCNA), a ring‐shaped homotrimer, is an essential factor in DNA metabolism including DNA replication and repair. PCNA functions in these reactions ...appear to be regulated by posttranslational modifications, such as ubiquitylation, sumoylation and
phosphorylation
. Recently, we have shown that PCNA phosphorylation by EGFR at tyrosine 211 (Y211) disrupts PCNA interactions with mismatch recognition proteins, thereby inhibiting DNA mismatch repair (MMR). In addition, phosphorylated PCNA promotes misincorporation during DNA replication. However, it is unknown whether one, two or all three phosphorylated subunits are responsible for MMR inhibition and error‐prone DNA synthesis. To answer this question, we created a series of phosphorylation‐mimicking and non‐phosphorylation‐mimicking PCNA molecules with variously phosphorylated subunits, and examined their roles in MMR and DNA synthesis
in vitro
and
in vivo
. Our biochemical experiments reveal that all forms of phosphorylated PCNA inhibit MMR, with the inhibition activity proportional to the increased levels of phosphorylation; all forms of phosphorylated PCNA slow down the speed of DNA synthesis while conducting error‐prone DNA synthesis. Our
in vivo
data show that high‐levels of PCNA phosphorylation induce cell death. This study therefore provides novel mechanisms for phosphorylated PCNA in cancer development and its potential role in cancer therapy.
Support or Funding Information
This work is supported in part by grants from National Natural Science Foundation of China (31370766), by Tsinghua‐Peking Joint Center for Life Sciences, and by National Institutes of Health of the United States (GM089684).
Tumors with defective mismatch repair (dMMR) are responsive to immunotherapy because of dMMR-induced neoantigens and activation of the cGAS-STING pathway. While neoantigens result from the ...hypermutable nature of dMMR, it is unknown how dMMR activates the cGAS-STING pathway. We show here that loss of the MutLα subunit MLH1, whose defect is responsible for ~50% of dMMR cancers, results in loss of MutLα-specific regulation of exonuclease 1 (Exo1) during DNA repair. This leads to unrestrained DNA excision by Exo1, which causes increased ssDNA formation, RPA exhaustion, DNA breaks, and aberrant DNA repair intermediates. Ultimately, this generates chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, activating the cGAS-STING pathway. In this study, we discovered a hitherto unknown MMR mechanism that modulates genome stability and has implications for cancer therapy.
The mechanism by which mismatch repair deficiency benefits immunotherapy is unclear. Guan et al. show that mismatch repair protein MLH1 controls Exo1 nuclease activity during DNA repair, and loss of MLH1 causes DNA hyper-excision, leading to chromosomal instability and cytosolic DNA accumulation. This activates the cGAS-STING pathway to facilitate immunotherapy.
During nuclear DNA replication, proofreading‐deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading‐proficient Pol δ or Pol ...ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα‐dependent, MutLα‐independent mismatch repair process we call Pol α‐segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5′ ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.
Synopsis
Okazaki fragment synthesis during eukaryotic DNA replication is initiated by proofreading‐deficient DNA polymerase α, requiring a dedicated repair process during Okazaki fragment maturation. This Pol alpha‐segment error editing (AEE) involves the exonuclease activity of flap endonuclease (FEN1) in cooperation with MutSα mismatch repair protein.
MutSα recognizes mismatches near the 5′ end of the DNA nick left after RNA primer flap cleavage.
MutSα interacts with FEN1 and stimulates mismatch removal by FEN1 exonuclease activity.
Defects in AEE lead to increased genome instability and cancer susceptibility.
The key role of proofreading‐deficient DNA polymerase α in initiating Okazaki fragment synthesis requires mismatch removal by a dedicated DNA repair process involving mammalian flap endonuclease.
Abstract only
The proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and repair, including DNA mismatch repair (MMR), an essential mechanism that ensures ...replication fidelity. PCNA is required for MMR at both the initiation and re‐synthesis steps. Recent studies have shown that PCNA is phosphorylated at tyrosine 211 (Y211) by epidermal growth factor receptor (EGFR), whose overexpression is associated with during tumor progression. We therefore hypothesize that the EGFR tumor‐promotion function is through PCNA Y211 phosphorylation by altering the MMR function. To test this hypothesis, we directly examined the influence of PCNA Y211 phosphorylation on MMR activity using a functional
in vitro
repair assay. We demonstrate here that nuclear extracts derived from tumor cells with high levels of phosphorylated PCNA are defective in MMR, and that the deficiency can be restored by purified non‐phosphorylated PCNA, suggesting that PCNA Y211 phosphorylation inhibits MMR. The inhibition was due to the inability of the phosphorylated PCNA to interact with mismatch recognition proteins MutSα and MutSβ. We also show that the PCNA‐phosphorylated nuclear extracts have a delayed DNA resynthesis during MMR, which is coupled with nucleotide misincorporations, leading to an elevated mutation frequency. Our study therefore demonstrates that a posttranslational modification promotes genome instability and tumor progression by negatively regulating the MMR function and DNA synthesis. Thus, the work provides a potential new biomarker for cancer progression.
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