Chronic exposure to arsenic is associated with the development of certain types of cancer, including lung and skin cancers. The exact mechanism by which arsenic induces tumorigenesis is unknown. ...Based on the fact that arsenic can generate reactive oxygen species to damage DNA and proteins, and that arsenic enhances expression of epidermal growth factor receptor (EGFR), which phosphorylates the proliferating cell nuclear antigen (PCNA), an important mismatch repair (MMR) component, we hypothesize that arsenic induces tumorigenesis is through its ability to inactivate the MMR system. We demonstrate here that exposure to arsenic indeed induces elevated EGFR expression and increased PCNA phosphorylation. Cells or cell extracts treated with arsenic are defective in MMR. Interestingly, the arsenic‐caused MMR deficiency can be reversed by PCNA, but not the PCNA preincubated with arsenic. We also found that the treatment of PCNA with arsenic changes PCNA conformation. These results suggest that arsenic inhibits MMR by directly altering the structure of PCNA, and by promoting PCNA phosphorylation through the activation of EGFR. Our work therefore reveals a novel mechanism by which arsenic induces carcinogenesis.
The human DNA repair enzyme MUTYH excises mispaired adenine residues in oxidized DNA. Homozygous MUTYH mutations underlie the autosomal, recessive cancer syndrome MUTYH-associated polyposis. We ...report a MUTYH variant, p.C306W (c.918C>G), with a tryptophan residue in place of native cysteine, that ligates the 4Fe4S cluster in a patient with colonic polyposis and family history of early age colon cancer. In bacterial MutY, the 4Fe4S cluster is redox active, allowing rapid localization to target lesions by long-range, DNA-mediated signalling. In the current study, using DNA electrochemistry, we determine that wild-type MUTYH is similarly redox-active, but MUTYH C306W undergoes rapid oxidative degradation of its cluster to 3Fe4S
, with loss of redox signalling. In MUTYH C306W, oxidative cluster degradation leads to decreased DNA binding and enzyme function. This study confirms redox activity in eukaryotic DNA repair proteins and establishes MUTYH C306W as a pathogenic variant, highlighting the essential role of redox signalling by the 4Fe4S cluster.
During nuclear DNA replication, proofreading-deficient DNA polymerase alpha (Pol alpha) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol ...delta or Pol epsi. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol alpha replication errors in a MutSalpha-dependent, MutLalpha-independent mismatch repair process we call Pol alpha-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 alpha, 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 MutSalpha mismatch repair protein. MutSalpha recognizes mismatches near the 5' end of the DNA nick left after RNA primer flap cleavage. MutSalpha interacts with FEN1 and stimulates mismatch removal by FEN1 exonuclease activity. Defects in AEE lead to increased genome instability and cancer susceptibility.
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.
MicroRNAs （miRNAs） are critical post-transcriptional regulators and are derived from hairpin-snaped 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 MutLa （MLH1-PMS2 heterodimer） leads to genome instability and tumorigenesis, but the mechanisms controlling MutLa 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 MutLa stimulates the conversion of pri-miRo422a to pre-miR-422a, as well as the processing of other miRNAs tested,
The proliferating cell nuclear antigen (PCNA) plays an important role in DNA replication and repair. Recent in vivo studies have shown that PCNA functions can be modulated by tyrosine211 (Tyr211) ...phosphorylation, leading to increase in cell proliferation and poor survival in some cancer patients. Since PCNA is required for DNA mismatch at the initiation and DNA re‐synthesis steps of DNA mismatch repair (MMR), we examined the effect of Tyr211‐phosphorylated PCNA on MMR. Tyr211‐phosphorylation and non‐phosphorylation mimic PCNAs were obtained and assayed for MMR in vitro. We demonstrate here that Tyr211‐phosphorylated PCNA blocks the in vitro MMR reaction at the initiation stage in both HeLa nuclear extract and the reconstituted MMR system, as little incision/excision product was detected in these reactions in the presence of Tyr211‐phosphorylated PCNA. However, in vitro gap‐filling and primer extension experiments suggest that the phosphorylated PCNA does not block DNA synthesis catalyzed by HeLa nuclear extract or pol δ. These observations suggest that Tyr211 phosphorylation regulates PCNA functions, possibly by promoting DNA replication but inhibiting DNA repair.
DNA mismatch repair (MMR) is a critical genome-maintenance system. It ensures genome stability by correcting mismatches generated during DNA replication, suppressing homologous recombination, and ...inducing apoptosis in response to severe DNA damage. As a result, defects in MMR lead to genome-wide mutations and susceptibility to both hereditary and sporadic cancer syndromes. The hallmark of cancer cells defective in MMR is their ability to display frequent instability in simple repetitive DNA sequences, a phenomenon called microsatellite instability (MSI). However, only ∼70% of the MSI-positive tumors have identifiable MMR gene mutations, indicating that additional factor(s) are responsible for the MSI phenotype in the remaining 30% MSI-tumors. We demonstrate here that phosphorylation of proliferating cell nuclear antigen (PCNA), an MMR component required for the initiation and resynthesis steps of the repair reactions, blocks in vitro MMR. We found that nuclear extracts derived from colorectal cell lines containing high levels of phosphorylated PCNA are not only defective in MMR, but also inhibitory to MMR activity in HeLa extracts. To determine if PCNA phosphorylation inhibits MMR, several PCNA isoforms that mimic phosphorylated or non-phosphorylated PCNA were examined for their effects on MMR activity. We show that all phosphorylated PCNA mimics block MMR at the initiation step but MMR was not affected by the non-phosphorylated mimetic PCNA. In vitro gap-filling experiments reveal that the phosphorylated PCNA induces a mutational frequency several fold higher than non-phosphorylated PCNA. Since PCNA has been shown to interact with MMR initiation factors MutSα and MutLα, we examined the interactions of phosphorylated PCNA with these two initiation factors. Interestingly, PCNA phosphorylation reduces the PCNA-MutSα interaction, but not the PCNA-MutLα interaction. Since PCNA is proposed to transfer MutSα to the mismatch site, the simplest explanation of the result is that PCNA phosphorylation inhibits MMR by blocking MutSα-mismatch binding activity. Taken together, our results reveal that PCNA phosphorylation induces genetic instability by inhibiting MMR at the initiation step and by promoting DNA polymerase-catalyzed mis-incorporations. This study provides a novel mechanism by which posttranslational modifications inhibit MMR, leading to genome instability and tumorigenesis. A second part of the study is to determine MMR function of several MutLα mutants associated with relapse leukemia patients. One of the mutants contains a phenylalanine99 to leucine substitution in the MLH1 subunit of MutLα. We show that this mutation inhibits MMR by blocking both the ATPase activity and the endonuclease activity associated with MutLα, supporting the importance of the MutLα ATPase and the endonuclease activities in MMR. KEYWORDS: PCNA, MutLα, Phosphorylation, HNPCC, AML.
DNA mismatch repair (MMR) is a critical genome-maintenance system. It ensures genome stability by correcting mismatches generated during DNA replication, suppressing homologous recombination, and ...inducing apoptosis in response to severe DNA damage. As a result, defects in MMR lead to genome-wide mutations and susceptibility to both hereditary and sporadic cancer syndromes. The hallmark of cancer cells defective in MMR is their ability to display frequent instability in simple repetitive DNA sequences, a phenomenon called microsatellite instability (MSI). However, only ~70% of the MSI-positive tumors have identifiable MMR gene mutations, indicating that additional factor(s) are responsible for the MSI phenotype in the remaining 30% MSI-tumors.
We demonstrate here that phosphorylation of proliferating cell nuclear antigen (PCNA), an MMR component required for the initiation and resynthesis steps of the repair reactions, blocks in vitro MMR. We found that nuclear extracts derived from colorectal cell lines containing high levels of phosphorylated PCNA are not only defective in MMR, but also inhibitory to MMR activity in HeLa extracts. To determine if PCNA phosphorylation inhibits MMR, several PCNA isoforms that mimic phosphorylated or non-phosphorylated PCNA were examined for their effects on MMR activity. We show that all phosphorylated PCNA mimics block MMR at the initiation step but MMR was not affected by the non-phosphorylated mimetic PCNA. In vitro gap-filling experiments reveal that the phosphorylated PCNA induces a mutational frequency several fold higher than non-phosphorylated PCNA. Since PCNA has been shown to interact with MMR initiation factors MutSα and MutLα, we examined the interactions of phosphorylated PCNA with these two initiation factors. Interestingly, PCNA phosphorylation reduces the PCNA-MutSα interaction, but not the PCNA-MutLα interaction. Since PCNA is proposed to transfer MutSα to the mismatch site, the simplest explanation of the result is that PCNA phosphorylation inhibits MMR by blocking MutSα-mismatch binding activity. Taken together, our results reveal that PCNA phosphorylation induces genetic instability by inhibiting MMR at the initiation step and by promoting DNA polymerase-catalyzed mis-incorporations. This study provides a novel mechanism by which posttranslational modifications inhibit MMR, leading to genome instability and tumorigenesis.
A second part of the study is to determine MMR function of several MutLα mutants associated with relapse leukemia patients. One of the mutants contains a phenylalanine99 to leucine substitution in the MLH1 subunit of MutLα. We show that this mutation inhibits MMR by blocking both the ATPase activity and the endonuclease activity associated with MutLα, supporting the importance of the MutLα ATPase and the endonuclease activities in MMR.
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