Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The oligonucleotide/oligosaccharide-binding (OB) fold is central to the architecture of single-stranded- DNA-binding proteins, which are polypeptides essential for diverse cellular processes, including DNA replication, repair, and recombination. In archaea, single-stranded DNA-binding proteins composed of multiple OB folds and a zinc finger domain, in a single polypeptide, have been described. The OB folds of these proteins were more similar to their eukaryotic counterparts than to their bacterial ones. Thus, the archaeal protein is called replication protein A (RPA), as in eukaryotes. Unlike most organisms, Methanosarcina acetivorans harbors multiple functional RPA proteins, and it was our interest to determine whether the different proteins play different roles in DNA transactions. Of particular interest was lagging-strand DNA synthesis, where recently RPA has been shown to regulate the size of the 5' region cleaved during Okazaki fragment processing. We report here that M. acetivorans RPA1 (MacRPA1), a protein composed of four OB folds in a single polypeptide, inhibits cleavage of a long flap (20 nucleotides) by M. acetivorans flap endonuclease 1 (MacFEN1). To gain a further insight into the requirement of the different regions of MacRPA1 on its inhibition of MacFEN1 endonuclease activity, N-terminal and C-terminal truncated derivatives of the protein were made and were biochemically and biophysically analyzed. Our results suggested that MacRPA1 derivatives with at least three OB folds maintained the properties required for inhibition of MacFEN1 endonuclease activity. Despite these interesting observations, further biochemical and genetic analyses are required to gain a deeper understanding of the physiological implications of our findings.
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PMID:Methanosarcina acetivorans flap endonuclease 1 activity is inhibited by a cognate single-stranded-DNA-binding protein. 1692 82

The mechanism of trinucleotide repeat expansion, an important cause of neuromuscular and neurodegenerative diseases, is poorly understood. We report here on the study of the role of flap endonuclease 1 (Fen1), a structure-specific nuclease with both 5' flap endonuclease and 5'-3' exonuclease activity, in the somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat of the DMPK gene in a mouse model for myotonic dystrophy type 1 (DM1). By intercrossing mice with Fen1 deficiency with transgenics with a DM1 (CTG)(n)*(CAG)(n) repeat (where 104n110), we demonstrate that Fen1 is not essential for faithful maintenance of this repeat in early embryonic cleavage divisions until the blastocyst stage. Additionally, we found that the frequency of somatic DM1 (CTG)(n)*(CAG)(n) repeat instability was essentially unaltered in mice with Fen1 haploinsufficiency up to 1.5 years of age. Based on these findings, we propose that Fen1, despite its role in DNA repair and replication, is not primarily involved in maintaining stability at the DM1 locus.
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PMID:Fen1 does not control somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat in a knock-in mouse model for DM1. 1697 12

Recently, we found an interaction between adenomatous polyposis coli (APC) and DNA polymerase beta (pol-beta) and showed that APC blocks strand-displacement synthesis of long-patch base excision repair (LP-BER) (Narayan, S., Jaiswal, A. S., and Balusu, R. (2005) J. Biol. Chem. 280, 6942-6949); however, the mechanism is not clear. Using an in vivo LP-BER assay system, we now show that the LP-BER is higher in APC-/- cells than in APC+/+ cells. In addition to pol-beta, the pull-down experiments showed that the full-length APC also interacted with flap endonuclease 1 (Fen-1). To further characterize the interaction of APC with pol-beta and Fen-1, we performed a domain-mapping of APC and found that both pol-beta and Fen-1 interact with a 138-amino acids peptide from the APC at the DRI-domain. Our functional assays showed that APC blocks pol-beta-mediated 1-nucleotide (1-nt) as well as strand-displacement synthesis of reduced abasic, nicked-, or 1-nt gapped-DNA substrates. Further studies demonstrated that APC blocks 5'-flap endonuclease as well as the 5'-3' exonuclease activity of Fen-1 resulting in the blockage of LP-BER. From these results, we concluded that APC can have three different effects on the LP-BER pathway. First, APC can block pol-beta-mediated 1-nt incorporation and strand-displacement synthesis. Second, APC can block LP-BER by blocking the coordinated formation and removal of the strand-displaced flap. Third, APC can block LP-BER by blocking hit-and-run synthesis. These studies will have important implications for APC in DNA damage-induced carcinogenesis and chemoprevention.
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PMID:Mechanism of adenomatous polyposis coli (APC)-mediated blockage of long-patch base excision repair. 1717 13

Trinucleotide repeat diseases, such as Huntington's disease, are caused by the expansion of trinucleotide repeats above a threshold of about 35 repeats. Once expanded, the repeats are unstable and tend to expand further both in somatic cells and during transmission, resulting in a more severe disease phenotype. Flap endonuclease 1 (Fen1), has an endonuclease activity specific for 5' flap structures and is involved in Okazaki fragment processing and base excision repair. Fen1 also plays an important role in preventing instability of CAG/CTG trinucleotide repeat sequences, as the expansion frequency of CAG/CTG repeats is increased in FEN1 mutants in vitro and in yeast cells defective for the yeast homolog, RAD27. Here we have tested whether one copy of yeast FEN1 is enough to maintain CAG/CTG tract stability in diploid yeast cells. We found that CAG/CTG repeats are stable in RAD27 +/- cells if the tract is 70 repeats long and exhibit a slightly increased expansion frequency if the tract is 85 or 130 repeats long. However for CAG-155 tracts, the repeat expansion frequency in RAD27 +/- cells is significantly higher than in RAD27 +/+ cells. This data indicates that cells containing longer CAG/CTG repeats need more Fen1 protein to maintain tract stability and that maintenance of long CAG/CTG repeats is particularly sensitive to Fen1 levels. Our results may explain the relatively small effects seen in the Huntington's disease (HD) FEN1 +/- heterozygous mice and myotonic dystrophy type 1 (DM1) FEN1 +/- heterozygous mice, and suggest that inefficient flap processing by Fen1 could play a role in the continued expansions seen in humans with trinucleotide repeat expansion diseases.
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PMID:Haploinsufficiency of yeast FEN1 causes instability of expanded CAG/CTG tracts in a length-dependent manner. 1738 31

Growing evidence suggests that the Rad9-Rad1-Hus1 complex (the 9-1-1 complex), besides its functions in DNA damage sensing and signaling pathways, plays also a direct role in various DNA repair processes. Recent studies have demonstrated that the 9-1-1 complex physically and functionally interacts with several components of the base excision repair (BER) machinery namely DNA polymerase beta (Pol beta), flap endonuclease 1 (Fen 1), DNA ligase I (Lig I) and the MutY homologue of Schizosaccharomyces pombe. In this work, we found for the first time that the 9-1-1 complex interacts in vitro and in vivo with the apurinic/apyrimidinic endonuclease 1 (APE 1), an early component of BER, and can stimulate its AP-endonuclease activity. Moreover, we show that the 9-1-1 complex possesses a stimulatory effect on long patch base excision repair (LP-BER) reconstituted in vitro. The enhancement of LP-BER activity is due to the specific stimulation of the two early components of the repair machinery, namely APE 1 and Pol beta, suggesting a hierarchy of interactions between the 9-1-1 complex and the BER proteins acting in the repairosome. Overall, our results indicate that the 9-1-1 complex is directly involved in LP-BER, thus providing a possible link between DNA damage checkpoints and BER.
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PMID:The checkpoint clamp, Rad9-Rad1-Hus1 complex, preferentially stimulates the activity of apurinic/apyrimidinic endonuclease 1 and DNA polymerase beta in long patch base excision repair. 1742 33

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5' nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5' arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5' arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5' arm, lies above a magnesium binding site. The less ordered 3' arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5' arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.
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PMID:Crystal structure of bacteriophage T4 5' nuclease in complex with a branched DNA reveals how flap endonuclease-1 family nucleases bind their substrates. 1769 99

Despite the wealth of information available on the biochemical functions and our recent findings of its roles in genome stability and cancer avoidance of the structure-specific flap endonuclease 1 (FEN1), its cellular compartmentalization and dynamics corresponding to its involvement in various DNA metabolic pathways are not yet elucidated. Several years ago, we demonstrated that FEN1 migrates into the nucleus in response to DNA damage and under certain cell cycle conditions. In the current paper, we found that FEN1 is superaccumulated in the nucleolus and plays a role in the resolution of stalled DNA replication forks formed at the sites of natural replication fork barriers. In response to UV irradiation and upon phosphorylation, FEN1 migrates to nuclear plasma to participate in the resolution of UV cross-links on DNA, most likely employing its concerted action of exonuclease and gap-dependent endonuclease activities. Based on yeast complementation experiments, the mutation of Ser(187)Asp, mimicking constant phosphorylation, excludes FEN1 from nucleolar accumulation. The replacement of Ser(187) by Ala, eliminating the only phosphorylation site, retains FEN1 in nucleoli. Both of the mutations cause UV sensitivity, impair cellular UV damage repair capacity, and decline overall cellular survivorship.
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PMID:Nucleolar localization and dynamic roles of flap endonuclease 1 in ribosomal DNA replication and damage repair. 1844 37

DNA alkylation-induced damage is one of the most efficacious anticancer therapeutic strategies. Enhanced DNA alkylation and weakened DNA repair capacity in cancer cells are responsible for the effectiveness of DNA-alkylating therapies. 5'-Flap endonuclease 1 (Fen1) is an important enzyme involved in base excision repair (BER), specifically in long-patch BER (LP-BER). Using the site-directed mutagenesis approach, we have identified an important role for amino acid Asp181 of Fen1 in its endonuclease activity. Asp181 is thought to be involved in Mg(2+) binding in the active site. Using structure-based molecular docking of Fen1 targeted to its metal binding pocket M2 (Mg(2+) site), we have identified a potent low-molecular weight inhibitor (LMI, NSC-281680) that efficiently blocks LP-BER. In this study, we have demonstrated that the interaction of this LMI with Fen1 blocked its endonuclease activity, thereby blocking LP-BER and enhancing the cytotoxic effect of DNA-alkylating agent Temozolomide (TMZ) in mismatch repair (MMR)-deficient and MMR-proficient colon cancer cells. The results further suggest that blockade of LP-BER by NSC-281680 may bypass other drug resistance mechanisms such as mismatch repair (MMR) defects. Therefore, our findings provide groundwork for the development of highly specific and safer structure-based small molecular inhibitors targeting the BER pathway, which can be used along with existing chemotherapeutic agents, like TMZ, as combination therapy for the treatment of colorectal cancer.
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PMID:Amino acid Asp181 of 5'-flap endonuclease 1 is a useful target for chemotherapeutic development. 1976 10

Flap endonuclease 1 (FEN1) and Dna2 endonuclease/helicase (Dna2) sequentially coordinate their nuclease activities for efficient resolution of flap structures that are created during the maturation of Okazaki fragments and repair of DNA damage. Acetylation of FEN1 by p300 inhibits its endonuclease activity, impairing flap cleavage, a seemingly undesirable effect. We now show that p300 also acetylates Dna2, stimulating its 5'-3' endonuclease, the 5'-3' helicase, and DNA-dependent ATPase activities. Furthermore, acetylated Dna2 binds its DNA substrates with higher affinity. Differential regulation of the activities of the two endonucleases by p300 indicates a mechanism in which the acetylase promotes formation of longer flaps in the cell at the same time as ensuring correct processing. Intentional formation of longer flaps mediated by p300 in an active chromatin environment would increase the resynthesis patch size, providing increased opportunity for incorrect nucleotide removal during DNA replication and damaged nucleotide removal during DNA repair. For example, altering the ratio between short and long flap Okazaki fragment processing would be a mechanism for better correction of the error-prone synthesis catalyzed by DNA polymerase alpha.
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PMID:Acetylation of Dna2 endonuclease/helicase and flap endonuclease 1 by p300 promotes DNA stability by creating long flap intermediates. 2001 87

Telomeres are terminal repetitive DNA sequences whose stability requires the coordinated actions of telomere-binding proteins and the DNA replication and repair machinery. Recently, we demonstrated that the DNA replication and repair protein Flap endonuclease 1 (FEN1) is required for replication of lagging strand telomeres. Here, we demonstrate for the first time that FEN1 is required for efficient re-initiation of stalled replication forks. At the telomere, we find that FEN1 depletion results in replicative stress as evidenced by fragile telomere expression and sister telomere loss. We show that FEN1 participation in Okazaki fragment processing is not required for efficient telomere replication. Instead we find that FEN1 gap endonuclease activity, which processes DNA structures resembling stalled replication forks, and the FEN1 interaction with the RecQ helicases are vital for telomere stability. Finally, we find that FEN1 depletion neither impacts cell cycle progression nor in vitro DNA replication through non-telomeric sequences. Our finding that FEN1 is required for efficient replication fork re-initiation strongly suggests that the fragile telomere expression and sister telomere losses observed upon FEN1 depletion are the direct result of replication fork collapse. Together, these findings suggest that other nucleases compensate for FEN1 loss throughout the genome during DNA replication but fail to do so at the telomere. We propose that FEN1 maintains stable telomeres by facilitating replication through the G-rich lagging strand telomere, thereby ensuring high fidelity telomere replication.
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PMID:FEN1 ensures telomere stability by facilitating replication fork re-initiation. 2055 83


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