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)

DNA double-strand break repair by homologous recombination entails nucleolytic resection of the 5' strand at break ends. Dna2, a flap endonuclease with 5'-3' helicase activity, is involved in the resection process. The Dna2 helicase activity has been implicated in Okazaki fragment processing during DNA replication but is thought to be dispensable for DNA end resection. Unexpectedly, we found a requirement for the helicase function of Dna2 in end resection in budding yeast cells lacking exonuclease 1. Biochemical analysis reveals that ATP hydrolysis-fueled translocation of Dna2 on ssDNA facilitates 5' flap cleavage near a single-strand-double strand junction while attenuating 3' flap incision. Accordingly, the ATP hydrolysis-defective dna2-K1080E mutant is less able to generate long products in a reconstituted resection system. Our study thus reveals a previously unrecognized role of the Dna2 translocase activity in DNA break end resection and in the imposition of the 5' strand specificity of end resection.
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PMID:A novel role of the Dna2 translocase function in DNA break resection. 2833 16

Human exonuclease 1 (hExo1) is a member of the RAD2/XPG structure-specific 5'-nuclease superfamily. Its dominant, processive 5'-3' exonuclease and secondary 5'-flap endonuclease activities participate in various DNA repair, recombination, and replication processes. A single active site processes both recessed ends and 5'-flap substrates. By initiating enzyme reactions in crystals, we have trapped hExo1 reaction intermediates that reveal structures of these substrates before and after their exo- and endonucleolytic cleavage, as well as structures of uncleaved, unthreaded, and partially threaded 5' flaps. Their distinctive 5' ends are accommodated by a small, mobile arch in the active site that binds recessed ends at its base and threads 5' flaps through a narrow aperture within its interior. A sequence of successive, interlocking conformational changes guides the two substrate types into a shared reaction mechanism that catalyzes their cleavage by an elaborated variant of the two-metal, in-line hydrolysis mechanism. Coupling of substrate-dependent arch motions to transition-state stabilization suppresses inappropriate or premature cleavage, enhancing processing fidelity. The striking reduction in flap conformational entropy is catalyzed, in part, by arch motions and transient binding interactions between the flap and unprocessed DNA strand. At the end of the observed reaction sequence, hExo1 resets without relinquishing DNA binding, suggesting a structural basis for its processivity.
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PMID:Interplay of catalysis, fidelity, threading, and processivity in the exo- and endonucleolytic reactions of human exonuclease I. 2853 82

Human exonuclease 1 (hEXO1) is a member of the 5'-nuclease superfamily and plays important roles in DNA repair. Along with acting as a 5'-exonuclease on blunt, gapped, nicked, and 3'-overhang DNAs, hEXO1 can also act as an endonuclease removing protruding 5'-single-stranded flaps from duplex ends. How hEXO1 and related 5'-nuclease human flap endonuclease 1 (hFEN1) are specific for discontinuous DNA substrates like 5'-flaps has been controversial. Here we report the first functional data that imply that hEXO1 threads the 5'-flap through a hole in the protein known as the helical arch, thereby excluding reactions of continuous single strands. Conjugation of bulky 5'-streptavidin that would "block" threading through the arch drastically slowed the hEXO1 reaction. In contrast, addition of streptavidin to a preformed hEXO1 5'-biotin flap DNA complex trapped a portion of the substrate in a highly reactive threaded conformation. However, another fraction behaves as if it were "blocked" and decayed very slowly, implying there were both threaded and unthreaded forms of the substrate present. The reaction of an unmodified hEXO1-flap DNA complex did not exhibit marked biphasic kinetics, suggesting a fast re-equilibration occurs that produces more threaded substrate when some decays. The finding that a threading mechanism like that used by hFEN1 is also used by hEXO1 unifies the mode of operation for members of the 5'-nuclease superfamily that act on discontinuous substrates. As with hFEN1, intrinsic disorder of the arch region of the protein may explain how flaps can be threaded without a need for a coupled energy source.
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PMID:Human Exonuclease 1 Threads 5'-Flap Substrates through Its Helical Arch. 2868 61

Enzymes of the 5' structure-specific nuclease family are crucial for DNA repair, replication, and recombination. One such enzyme is the human exonuclease 1 (hExo1) metalloenzyme, which cleaves DNA strands, acting primarily as a processive 5'-3' exonuclease and secondarily as a 5'-flap endonuclease. Recently, in crystallo reaction intermediates have elucidated how hExo1 exerts hydrolysis of DNA phosphodiester bonds. These hExo1 structures show a third metal ion intermittently bound close to the two-metal-ion active site, to which recessed ends or 5'-flap substrates bind. Evidence of this third ion has been observed in several nucleic-acid-processing metalloenzymes. However, there is still debate over what triggers the (un)binding of this transient third ion during catalysis and whether this ion has a catalytic function. Using extended molecular dynamics and enhanced sampling free-energy simulations, we observed that the carboxyl side chain of Glu89 (located along the arch motif in hExo1) flips frequently from the reactant state to the product state. The conformational flipping of Glu89 allows one metal ion to be recruited from the bulk and promptly positioned near the catalytic center. This is in line with the structural evidence. Additionally, our simulations show that the third metal ion assists the departure, through the mobile arch, of the nucleotide monophosphate product from the catalytic site. Structural comparisons of nuclease enzymes suggest that this Glu(Asp)-mediated mechanism for third ion recruitment and nucleic acid hydrolysis may be shared by other 5' structure-specific nucleases.
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PMID:Recruiting Mechanism and Functional Role of a Third Metal Ion in the Enzymatic Activity of 5' Structure-Specific Nucleases. 3193 91


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