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)

Human Proliferating Cellular Nuclear Antigen (hPCNA), a member of the sliding clamp family of proteins, makes specific protein-protein interactions with DNA replication and repair proteins through a small peptide motif termed the PCNA-interacting protein, or PIP-box. We solved the structure of hPCNA bound to PIP-box-containing peptides from the p66 subunit of the human replicative DNA polymerase-delta (452-466) at 2.6 A and of the flap endonuclease (FEN1) (331-350) at 1.85 A resolution. Both structures demonstrate that the pol-delta p66 and FEN1 peptides interact with hPCNA at the same site shown to bind the cdk-inhibitor p21(CIP1). Binding studies indicate that peptides from the p66 subunit of the pol-delta holoenzyme and FEN1 bind hPCNA from 189- to 725-fold less tightly than those of p21. Thus, the PIP-box and flanking regions provide a small docking peptide whose affinities can be readily adjusted in accord with biological necessity to mediate the binding of DNA replication and repair proteins to hPCNA.
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PMID:Structural and thermodynamic analysis of human PCNA with peptides derived from DNA polymerase-delta p66 subunit and flap endonuclease-1. 1557 34

During lagging strand DNA replication, the Okazaki fragment maturation machinery is required to degrade the initiator RNA with high speed and efficiency, and to generate with great accuracy a proper DNA nick for closure by DNA ligase. Several operational parameters are important in generating and maintaining a ligatable nick. These are the strand opening capacity of the lagging strand DNA polymerase delta (Pol delta ), and its ability to limit strand opening to that of a few nucleotides. In the presence of the flap endonuclease FEN1, Pol delta rapidly hands off the strand-opened product for cutting by FEN1, while in its absence, the ability of DNA polymerase delta to switch to its 3'-->5'-exonuclease domain in order to degrade back to the nick position is important in maintaining a ligatable nick. This regulatory system has a built-in redundancy so that dysfunction of one of these activities can be tolerated in the cell. However, further dysfunction leads to uncontrolled strand displacement synthesis with deleterious consequences, as is revealed by genetic studies of exonuclease-defective mutants of S. cerevisiae Pol delta. These same parameters are also important for other DNA metabolic processes, such as base excision repair, that depend on Pol delta for synthesis.
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PMID:How the cell deals with DNA nicks. 1565 50

Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase ot-primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase ca and recruits DNA polymerase S and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase 8, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase E normally replicates this strand,but under conditions of dysfunction, DNA polymerase 8 may substitute.
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PMID:DNA polymerases that propagate the eukaryotic DNA replication fork. 1581 31

In response to DNA damage, the Rad6/Rad18 ubiquitin-conjugating complex monoubiquitinates the replication clamp proliferating cell nuclear antigen (PCNA) at Lys-164. Although ubiquitination of PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast PCNA specifically on Lys-164. Significantly, only those PCNA clamps that are appropriately loaded around effector DNA by its loader, replication factor C, are ubiquitinated. This observation suggests that, in vitro, only PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated PCNA displays the same replicative functions as unmodified PCNA. These functions include loading onto DNA by replication factor C, as well as Okazaki fragment synthesis and maturation by the PCNA-coordinated actions of DNA polymerase delta, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase zeta remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase eta, the yeast Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.
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PMID:Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases eta and REV1. 1634 68

The archaeal/eukaryotic proliferating cell nuclear antigen (PCNA) toroidal clamp interacts with a host of DNA modifying enzymes, providing a stable anchorage and enhancing their respective processivities. Given the broad range of enzymes with which PCNA has been shown to interact, relatively little is known about the mode of assembly of functionally meaningful combinations of enzymes on the PCNA clamp. We have determined the X-ray crystal structure of the Sulfolobus solfataricus PCNA1-PCNA2 heterodimer, bound to a single copy of the flap endonuclease FEN1 at 2.9 A resolution. We demonstrate the specificity of interaction of the PCNA subunits to form the PCNA1-PCNA2-PCNA3 heterotrimer, as well as providing a rationale for the specific interaction of the C-terminal PIP-box motif of FEN1 for the PCNA1 subunit. The structure explains the specificity of the individual archaeal PCNA subunits for selected repair enzyme 'clients', and provides insights into the co-ordinated assembly of sequential enzymatic steps in PCNA-scaffolded DNA repair cascades.
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PMID:Structure of an archaeal PCNA1-PCNA2-FEN1 complex: elucidating PCNA subunit and client enzyme specificity. 1694 55

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

Proliferating cell nuclear antigen (PCNA) is a well-known multifunctional protein involved in eukaryotic and archaeal DNA transactions. The homotrimeric PCNA ring encircles double-stranded DNA within its central hole and tethers many proteins on DNA. Plural genes encoding PCNA-like proteins have been found in the genome sequence of crenarchaeal organisms. We describe here the biochemical properties of the three PCNAs, PCNA1, PCNA2 and PCNA3, from the hyperthermophilic archaeon, Aeropyrum pernix. PCNA2 can form a trimeric structure by itself, and it also forms heterotrimeric structures with PCNA1 and PCNA3. However, neither PCNA1 nor PCNA3 can form homotrimers. The DNA synthesis activity of DNA polymerase I and II, the endonuclease activity of FEN1, and the nick-sealing activity of DNA ligase were stimulated by the complex of PCNA2 and 3 or PCNA1, 2 and 3. These results suggest that the heterotrimeric PCNA at least including PCNA2 and 3 function as the clamp in the replisome. However, PCNA2 is the most abundant in the cells throughout the growth stages among the three PCNAs, and therefore, PCNA2 may perform multitasks by changing complex composition.
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PMID:Specific interactions of three proliferating cell nuclear antigens with replication-related proteins in Aeropyrum pernix. 1749 21

Deoxyribose phosphate (dRP) removal by DNA polymerase beta (Pol beta) is a pivotal step in base excision repair (BER). To identify BER cofactors, especially those with dRP lyase activity, we used a Pol beta null cell extract and BER intermediate as bait for sodium borohydride crosslinking. Mass spectrometry identified the high-mobility group box 1 protein (HMGB1) as specifically interacting with the BER intermediate. Purified HMGB1 was found to have weak dRP lyase activity and to stimulate AP endonuclease and FEN1 activities on BER substrates. Coimmunoprecipitation experiments revealed interactions of HMGB1 with known BER enzymes, and GFP-tagged HMGB1 was found to accumulate at sites of oxidative DNA damage in living cells. HMGB1(-/-) mouse cells were slightly more resistant to MMS than wild-type cells, probably due to the production of fewer strand-break BER intermediates. The results suggest HMGB1 is a BER cofactor capable of modulating BER capacity in cells.
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PMID:HMGB1 is a cofactor in mammalian base excision repair. 1780 46

Base excision repair (BER) corrects a variety of small base lesions in DNA. The UNG gene encodes both the nuclear (UNG2) and the mitochondrial (UNG1) forms of the human uracil-DNA glycosylase (UDG). We prepared mitochondrial extracts free of nuclear BER proteins from human cell lines. Using these extracts we show that UNG is the only detectable UDG in mitochondria, and mitochondrial BER (mtBER) of uracil and AP sites occur by both single-nucleotide insertion and long-patch repair DNA synthesis. Importantly, extracts of mitochondria carry out repair of modified AP sites which in nuclei occurs through long-patch BER. Such lesions may be rather prevalent in mitochondrial DNA because of its proximity to the electron transport chain, the primary site of production of reactive oxygen species. Furthermore, mitochondrial extracts remove 5' protruding flaps from DNA which can be formed during long-patch BER, by a "flap endonuclease like" activity, although flap endonuclease (FEN1) is not present in mitochondria. In conclusion, combined short- and long-patch BER activities enable mitochondria to repair a broader range of lesions in mtDNA than previously known.
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PMID:Mitochondrial base excision repair of uracil and AP sites takes place by single-nucleotide insertion and long-patch DNA synthesis. 1829 53

DNA tandem lesions are comprised of two contiguously damaged nucleotides. This subset of clustered lesions is produced by a variety of oxidizing agents, including ionizing radiation. Clustered lesions can inhibit base excision repair (BER). We report the effects of tandem lesions composed of a thymine glycol and a 5'-adjacent 2-deoxyribonolactone (LTg) or tetrahydrofuran abasic site (FTg). Some BER enzymes that act on the respective isolated lesions do not accept the tandem lesion as a substrate. For instance, endonuclease III (Nth) does not excise thymine glycol (Tg) when it is part of either tandem lesion. Similarly, endonuclease IV (Nfo) does not incise L or F when they are in tandem with Tg. Long-patch BER overcomes inhibition by the tandem lesion. DNA polymerase beta (Pol beta) carries out strand displacement synthesis, following APE1 incision of the abasic site. Pol beta activity is enhanced by flap endonuclease (FEN1), which cleaves the resulting flap. The tandem lesion is also incised by the bacterial nucleotide excision repair system UvrABC with almost the same efficiency as an isolated Tg. These data reveal two solutions that DNA repair systems can use to counteract the formation of tandem lesions.
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PMID:DNA tandem lesion repair by strand displacement synthesis and nucleotide excision repair. 1834 Dec 93


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