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)

Blockage of replication fork progression often occurs during DNA replication, and repairing and restarting stalled replication forks are essential events in all organisms for the maintenance of genome integrity. The repair system employs processing enzymes to restore the stalled fork. In Archaea Hef is a well conserved protein that specifically cleaves nicked, flapped, and fork-structured DNAs. This enzyme contains two distinct domains that are similar to the DEAH helicase family and XPF nuclease superfamily proteins. Analyses of truncated mutant proteins consisting of each domain revealed that the C-terminal nuclease domain independently recognized and incised fork-structured DNA. The N-terminal helicase domain also specifically unwound fork-structured DNA and Holliday junction DNA in the presence of ATP. Moreover, the endonuclease activity of the whole Hef protein was clearly stimulated by ATP hydrolysis catalyzed by the N-terminal domain. These enzymatic properties suggest that Hef efficiently resolves stalled replication forks by two steps, which are branch point transfer to the 5'-end of the nascent lagging strand by the N-terminal helicase followed by template strand incision for leading strand synthesis by the C-terminal endonuclease.
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PMID:Cooperation of the N-terminal Helicase and C-terminal endonuclease activities of Archaeal Hef protein in processing stalled replication forks. 1548 82

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. The xeroderma pigmentosum group B (XPB) helicase subunit of TFIIH functions in NER and transcription. The serine 751 (S751) residue of XPB was found to be phosphorylated in vivo. This phosphorylation inhibits NER and the microinjection of a phosphomimicking XPB-S751E mutant is unable to correct the NER defect of XP-B cells. Conversely, XPB-S751 dephosphorylation or its substitution with alanine (S751A) restores NER both in vivo and in vitro. Surprisingly, phospho/dephosphorylation of S751 spares TFIIH-dependent transcription. Finally, the phosphorylation of XPB-S751 does not impair the TFIIH unwinding of the DNA around the lesion, but rather prevents the 5' incision triggered by the ERCC1-XPF endonuclease. These data support an additional role for XPB in promoting the incision of the damaged fragment and reveal a point of NER regulation on TFIIH without interference in its transcription activity.
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PMID:Phosphorylation of XPB helicase regulates TFIIH nucleotide excision repair activity. 1554 33

Structure-specific nucleases of the XPF/Mus81 family function in several DNA recombination and repair pathways in eukaryotes, cleaving a variety of flap and branched DNA substrates. Mus81 and XPF are clearly related evolutionarily but differ markedly in their substrate specificity and protein partners. We demonstrate that the XPF endonuclease from Sulfolobus solfataricus, which is dependent on the sliding clamp proliferating cell nuclear antigen for activity, represents an ancestral form of the XPF/Mus81 family, with key properties in common with both enzymes. The archaeal XPF has a domain organization and sequence preference very similar to eukaryal XPF-ERCC1. However, the archaeal enzyme has a pronounced preference for Mus81-type substrates such as D loops, nicked four-way junctions, and 3' flaps. These all have in common a 5'-DNA end next to the cleavage site. The availability of the sliding clamp proliferating cell nuclear antigen may dictate the activity of Sulfolobus XPF in vivo.
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PMID:An archaeal endonuclease displays key properties of both eukaryal XPF-ERCC1 and Mus81. 1559 Oct 65

The genetically complex disease Fanconi anemia (FA) comprises cancer predisposition, developmental defects, and bone marrow failure due to elevated apoptosis. The FA cellular phenotype includes universal sensitivity to DNA crosslinking damage, symptoms of oxidative stress, and reduced mutability at the X-linked HPRT gene. In this review article, we present a new heuristic molecular model that accommodates these varied features of FA cells. In our view, the FANCA, -C, and -G proteins, which are both cytoplasmic and nuclear, have an integrated dual role in which they sense and convey information about cytoplasmic oxidative stress to the nucleus, where they participate in the further assembly and functionality of the nuclear core complex (NCCFA= FANCA/B/C/E/F/G/L). In turn, NCCFA facilitates DNA replication at sites of base damage and strand breaks by performing the critical monoubiquitination of FANCD2, an event that somehow helps stabilize blocked and broken replication forks. This stabilization facilitates two kinds of processes: translesion synthesis at sites of blocking lesions (e.g., oxidative base damage), which produces point mutations by error-prone polymerases, and homologous recombination-mediated restart of broken forks, which arise spontaneously and when crosslinks are unhooked by the ERCC1-XPF endonuclease. In the absence of the critical FANCD2 monoubiquitination step, broken replication forks further lose chromatid continuity by collapsing into a configuration that is more difficult to restart through recombination and prone to aberrant repair through nonhomologous end joining. Thus, the FA regulatory pathway promotes chromosome integrity by monitoring oxidative stress and coping efficiently with the accompanying oxidative DNA damage during DNA replication.
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PMID:How Fanconi anemia proteins promote the four Rs: replication, recombination, repair, and recovery. 1566 41

The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1-/- mice was compared to that in young and old wild-type mice. Ercc1-/- mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1-/- mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa-/- mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging.
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PMID:Reduced hematopoietic reserves in DNA interstrand crosslink repair-deficient Ercc1-/- mice. 1569 71

Human XPF-ERCC1 is a DNA endonuclease that incises a damaged DNA strand on the 5' side of a lesion during nucleotide excision repair and has additional role(s) in homologous recombination and DNA interstrand crosslink repair. We show that a truncated form of XPF lacking the N-terminal helicase-like domain in complex with ERCC1 exhibits a structure-specific endonuclease activity with similar specificity to that of full-length XPF-ERCC1. Two domains of ERCC1, a central domain and a C-terminal tandem helix-hairpin-helix (HhH2) dimerization domain, bind to ssDNA. The central domain of ERCC1 binds ssDNA/dsDNA junctions with a defined polarity, preferring a 5' single-stranded overhang. The XPF-ERCC1 HhH2 domain heterodimer contains two independent ssDNA-binding surfaces, which are revealed by a crystal structure of the protein complex. A crystal structure of the central domain of ERCC1 shows its fold is strikingly similar to that of the nuclease domains of the archaeal Mus81/XPF homologs, despite very low sequence homology. A groove lined with basic and aromatic residues on the surface of ERCC1 has apparently been adapted to interact with ssDNA. On the basis of these crystallographic and biochemical studies, we propose a model in which XPF-ERCC1 recognizes a branched DNA substrate by binding the two ssDNA arms with the two HhH2 domains of XPF and ERCC1 and by binding the 5'-ssDNA arm with the central domain of ERCC1.
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PMID:Crystal structure and DNA binding functions of ERCC1, a subunit of the DNA structure-specific endonuclease XPF-ERCC1. 1607 55

The XPF/Mus81 family of structure-specific nucleases cleaves branched or nicked DNA substrates and are implicated in a wide range of DNA repair and recombination processes. The structure of the crenarchaeal XPF bound to a DNA duplex has revealed a plausible mechanism for DNA binding, involving DNA distortion into upstream and downstream duplexes engaged by the two helix-hairpin-helix domains that form a dimeric structure at the C-terminus of the enzyme. A flexible linker joins these to the dimeric nuclease domain, and a C-terminal motif interacts with the sliding clamp, which is essential for the activity of the enzyme. Here, we demonstrate the importance of the downstream duplex in directing the endonuclease activity of crenarchaeal XPF, which is similar to that of Mus81-Eme1, and suggest a mechanistic basis for this control. Furthermore, our data reveal that the enzyme can digest a nicked DNA strand processively over at least 60 nt in a 3'-5' direction and can remove varied types of DNA lesions and blocked DNA termini. This in vitro activity suggests a potential role for crenarchaeal XPF in a variety of repair processes for which there are no clear pathways in archaea.
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PMID:DNA end-directed and processive nuclease activities of the archaeal XPF enzyme. 1631 25

The human ERCC1/XPF complex is a structure-specific endonuclease with defined polarity that participates in multiple DNA repair pathways. We report the heterodimeric structure of the C-terminal domains of both proteins responsible for ERCC1/XPF complex formation. Both domains exhibit the double helix-hairpin-helix motif (HhH)2, and they are related by a pseudo-2-fold symmetry axis. In the XPF domain, the hairpin of the second motif is replaced by a short turn. The ERCC1 domain folds properly only in the presence of the XPF domain, which implies a role for XPF as a scaffold for the folding of ERCC1. The intersubunit interactions are largely hydrophobic in nature. NMR titration data show that only the ERCC1 domain of the ERCC1/XPF complex is involved in DNA binding. On the basis of these findings, we propose a model for the targeting of XPF nuclease via ERCC1-mediated interactions in the context of nucleotide excision repair.
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PMID:The structure of the human ERCC1/XPF interaction domains reveals a complementary role for the two proteins in nucleotide excision repair. 1633 1

The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3' side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5' incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.
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PMID:Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced dna damage depends on functional TFIIH. 1700 Jul 69

TRF2, a telomere-binding protein, is a crucial player in telomere length maintenance. Overexpression of TRF2 results in telomere shortening in both normal primary fibroblasts and telomerase-positive cancer cells. TRF2 is found to be associated with XPF-ERCC1, a structure-specific endonuclease involved in nucleotide excision repair, crosslink repair and DNA recombination. XPF-ERCC1 is implicated in TRF2-dependent telomere loss in mouse keratinocytes, however, whether XPF-ERCC1 and its nuclease activity are required for TRF2-mediated telomere shortening in human cells is unknown. Here we report that TRF2-induced telomere shortening is abrogated in human cells deficient in XPF, demonstrating that XPF-ERCC1 is required for TRF2-promoted telomere shortening. To further understand the role of XPF in TRF2-dependent telomere shortening, we generated constructs containing either wild type XPF or mutant XPF proteins carrying amino acid substitutions in its conserved nuclease domain. We show that wild type XPF can complement XPF-deficient cells for repair of UV-induced DNA damage whereas the nuclease-inactive XPF proteins fail to do so, indicating that the nuclease activity of XPF is essential for nucleotide excision repair. In contrast, both wild type XPF and nuclease-inactive XPF proteins, when expressed in XPF-deficient cells, are able to rescue TRF2-mediated telomere shortening. Thus, our results suggest that the function of XPF in TRF2-mediated telomere shortening is conserved between mouse and human. Furthermore, our findings reveal an unanticipated nuclease-independent function of XPF in TRF2-mediated telomere shortening.
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PMID:XPF with mutations in its conserved nuclease domain is defective in DNA repair but functions in TRF2-mediated telomere shortening. 1705 45


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