Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.1 (S1 nuclease)
 3,660 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The RecBCD-K177Q enzyme has a lysine-to-glutamine mutation in the putative ATP-binding sequence of the RecD protein (Korangy, F., and Julin, D.A. (1992) J. Biol. Chem. 267, 1727-1732). We have compared the enzymatic properties of the RecBCD-K177Q enzyme with those of the wild-type RecBCD enzyme from Escherichia coli. The purified RecBCD-K177Q enzyme has ATP-dependent nuclease activity on double-stranded or denatured DNA which is reduced (4-14-fold less) compared with the wild type. The kcat and Km(ATP) for ATP hydrolysis stimulated by double-stranded DNA are both reduced in RecBCD-K177Q, so that kcat/Km(ATP) is relatively unaffected. The mutant enzyme is impaired in its ability to unwind DNA in an assay where single-stranded DNA is trapped by the single-stranded DNA binding protein and subsequently degraded by S1 nuclease. The mutant enzyme also produces fewer acid-soluble DNA nucleotides per ATP hydrolyzed than does the wild type, at low ATP concentrations (less than 20 microM).
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PMID:Enzymatic effects of a lysine-to-glutamine mutation in the ATP-binding consensus sequence in the RecD subunit of the RecBCD enzyme from Escherichia coli. 130 93

Modification of the histidine residues of purified S1 nuclease resulted in loss of its single-stranded (ss)DNAase, RNAase and phosphomonoesterase activities. Kinetics of inactivation indicated the involvement of a single histidine residue in the catalytic activity of the enzyme. Furthermore, histidine modification was accompanied by the concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of ssDNA, RNA and 3'-AMP. Substrate protection was not observed against Methylene Blue- and diethyl pyrocarbonate (DEP)-mediated inactivation. The histidine (DEP)-modified enzyme could effectively bind 5'-AMP, a competitive inhibitor of S1 nuclease, whereas the lysine (2,4,6-trinitrobenzenesulphonic acid)-modified enzyme showed a significant decrease in its ability to bind 5'-AMP. The inability of the substrates to protect the enzyme against DEP-mediated inactivation, coupled with the ability of the modified enzyme to bind 5'-AMP effectively, suggests the involvement of histidine in catalysis.
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PMID:Active-site characterization of S1 nuclease. II. Involvement of histidine in catalysis. 146 60

A simple procedure, involving heat-treatment, DEAE-Sephadex, AMP-Sepharose and Bio-Gel P-60 chromatography, was developed for the purification of S1 nuclease to homogeneity from commercially available Takadiastase powder. Chemical modification of the amino groups of purified S1 nuclease revealed that lysine is essential for single-stranded DNAase, RNAase and phosphomonoesterase activities associated with the enzyme. The kinetics of inactivation suggested the involvement of a single lysine residue in the active site of the enzyme. Additionally, lysine modification was accompanied by a concomitant loss of all the activities of the enzyme, indicating the presence of a common catalytic site responsible for the hydrolysis of single-stranded DNA, RNA and 3'-AMP. Substrate-protection and inhibitor-binding studies on enzyme modified with 2,4,6-trinitrobenzenesulphonic acid showed that lysine may be involved in the substrate binding.
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PMID:Active-site characterization of S1 nuclease. I. Affinity purification and influence of amino-group modification. 163 40

On in vitro transcription of total genomic DNA of the tortoise (Geoclemys reevessi), a discrete-sized RNA of 6.5S was obtained that represented a highly repetitive and transcribable sequence in the tortoise genome. Three sequences of the 6.5S RNA gene were sequenced, and a consensus sequence was deduced from these three sequences and one reported previously [Endoh, H & Okada, N. (1986) Proc. Natl Acad. Sci. USA 83, 251-255]. The 5' part of the gene showed close similaries to lysine (rabbit) and threonine (mouse) tRNAs (overall similarity 68-70%), so this tortoise sequence may have evolved from one of these tRNAs. The consensus sequence retained the expected CCA triplet at the 3' end of tRNA, but not at the 3' end of tDNA, supporting the idea that the tRNA-related region of the gene was generated via an RNA intermediate. The 5' and 3' flanking sequences of the four genes were found to be completely different from each other. Fingerprint analysis and S1 nuclease mapping analysis also showed that sequence boundaries of tortoise repetitive units exactly corresponded to RNA species. These results, together with data obtained by Southern blot hybridization, indicated that the 6.5S RNA genes are dispersed in the tortoise genome. Therefore, generation of the tRNA-related region of the gene and amplification of the whole unit of the gene are both RNA-mediated events. The existence of this tortoise sequence suggests that short interspersed sequences are more common in eukaryotic genomes than had previously been thought.
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PMID:A highly repetitive and transcribable sequence in the tortoise genome is probably a retroposon. 169 79

The LYS5 and LYS2 genes of Saccharomyces cerevisiae are required for the synthesis of alpha-aminoadipate reductase in the lysine pathway. The LYS5 gene, originally cloned as a DNA insert of the plasmid pSC5, has been subcloned on a 3.2 kb SphI-Sau3AI DNA fragment of the recombinant plasmid pSR7. An internal 2.1 kb HpaI-HpaI DNA fragment of the subclone, upon Southern hybridization, exhibits homology with HpaI-restricted wild-type S. cerevisiae genomic DNA. The lys5+ transformants exhibited alpha-aminoadipate reductase activity similar to that of wild-type cells. S1 nuclease analysis localizes the transcription initiation site relative to the detailed restriction map, and reveals the direction of transcription, as well as the transcript size of the LYS5 gene which can be no greater than 1.65 kb. From this it is estimated that the encoded polypeptide is appreciably smaller than the 4 kb LYS2 gene product. These results provide a physical and biochemical characterization of the cloned LYS5 gene. Based on these observations, it is concluded that the LYS5 gene encodes a relatively small polypeptide of the large heteropolymeric alpha-aminoadipate reductase.
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PMID:Physical and biochemical characterization of the cloned LYS5 gene required for alpha-aminoadipate reductase activity in the lysine biosynthetic pathway of Saccharomyces cerevisiae. 173 23

Maximum expression of the Corynebacterium glutamicum lysA gene is dependent upon the presence of a 2.3 kb region immediately 5' of the lysA reading frame. Subcloning and functional analysis of the upstream region implied that this region contained the lysA promoter. Sequence determination of the upstream region revealed a single open reading frame, orfX, in the same orientation as lysA. The orfX coding sequence exhibited all the sequence characteristics of a gene with the potential for a 550-amino-acid polypeptide product. Expression of lysA is coupled to that of orfX via a common promoter located immediately 5' of orfX. The RNA start site has been determined by S1 nuclease mapping. Both the orfX and the lysA gene are expressed as a single 3.0 kb RNA transcript. These data indicate that orfX and lysA are genes within a two-gene operon. Expression of the lysA gene is not subject to regulation by lysine. The orfX gene product was shown not to be directly linked to the lysine biosynthetic pathway, nor is it the enzyme incorporating DAP into the peptidoglycan precursor.
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PMID:Nucleotide sequence and organization of the upstream region of the Corynebacterium glutamicum lysA gene. 849 94

The complete nucleotide sequences of the 1.2-kilobase HindIII fragments which contain the pilin genes of two independently isolated strains of Pseudomonas aeruginosa (PAK and PA103) have been determined and compared to that of strain PA01 (Sastry, P. A., Finlay, B. B., Pasloske, B. L., Paranchych, W., Pearlstone, J. R., and Smollier, L. B. (1985) J. Bacteriol. 164, 571-577). The fragments share extensive regions of homology, including the 5'- and 3'-flanking sequences as well as the 5' end of the pilin gene. The most highly diverged segments of the pilin genes are those which encode the variable carboxyl-terminal region of the pilin polypeptides. The pilin polypeptides each contain a 6-amino acid amino-terminal leader peptide (Met-Lys-Ala-Gln-Lys-Gly) and are nearly identical in the following 60 amino acids. The carboxyl-terminal portion of the pilin polypeptides contain extensive regions of divergence in their amino acid sequences, although hydropathicity analysis of the pilin polypeptides indicated that they are structurally similar. The transcriptional initiation site of the PAK pilin gene has been determined by S1 nuclease mapping. The promoter region at -10 and -35 base pairs from the transcriptional initiation site shows no significant homology to the consensus Escherichia coli promoter, but the -12 and -24 regions show a high degree of homology to promoters which require the ntrA gene product for transcription. Several other Pseudomonas promoters and the promoters of the homologous pilin genes from other bacterial species also share homology to this sequence.
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PMID:Nucleotide sequence and transcriptional initiation site of two Pseudomonas aeruginosa pilin genes. 243 Sep 61

Neuronal cells express a pp60c-src variant that displays an altered electrophoretic mobility and a different V8 peptide pattern relative to pp60c-src expressed in tissues of non-neuronal origin. To determine whether the neuronal form of pp60c-src is encoded by a brain-specific messenger RNA, a mouse brain complementary DNA (cDNA) library was screened with a chicken c-src probe and a 3.8-kilobase c-src cDNA clone was isolated. This clone encodes a 60-kilodalton protein that differs from chicken or human pp60c-src primarily in having six extra amino acids (Arg-Lys-Val-Asp-Val-Arg) within the NH2-terminal 16 kilodaltons of the molecule. S1 nuclease protection analysis confirmed that brain c-src RNA contains an 18-nucleotide insertion at the position of the extra six amino acids. This insertion occurs at a position that corresponds to a splice junction in the chicken and human c-src genes. The isolated c-src cDNA clone encodes a protein that displays an identical V8 peptide pattern to that observed in pp60c-src isolated from tissues of neuronal origin.
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PMID:Neuronal pp60c-src contains a six-amino acid insertion relative to its non-neuronal counterpart. 244 Jan 6

Agarose-poly-L-lysine (Ag-(lys)n-DNA) has been used to bind DNA for assay of anti-DNA antibodies (ab). In this work, an algorithmic approach has been used to classify antinuclear ab (ANA) as being directed against native DNA (dsDNA), denatured DNA (ssDNA), DNA-protein complexes (deoxyribonucleoprotein; DNP), and against antigens which are independent of DNA (iDNA). These ab were subjected to Ag-(lys)n-DNA, and the selectivity of this adsorbent for the various specificities of ab was determined. The DNA on the columns was left untreated or treated with S1 nuclease, this being effected either by treating the DNA prior to introducing it onto the columns or by adding S1 nuclease to the columns after the DNA was bound. Ag-(lys)n-DNA adsorbs ab directed against ssDNA and DNP as well as ab to dsDNA; iDNA ab are not adsorbed. S1 nuclease treatment does not effectively remove ssDNA regions from the Ag-(lys)n-DNA, but it does result in the abolition of the adsorption of a population of ab which are in the anti-DNP sera and contribute to the total ANA load. While anti-iDNA ab are not adsorbed onto the columns, they do contribute to the ANA titer, unlike anti-ssDNA ab which are adsorbed onto the Ag-(lys)n-DNA but do not contribute to the ANA titer. We conclude that Ag-(lys)n-DNA bears antigenic sites for dsDNA, ssDNA, and DNP ab and suggest that our understanding of the characteristic ab-binding profile of this versatile immunoadsorbent may have applications in the study of autoimmune diseases.
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PMID:Characteristics of antibodies adsorbed on the DNA immunoadsorbent, agarose poly-L-lysine-DNA. 244 98

The nucleotide sequence of the human spumaretrovirus (HSRV) genome was determined. The 5' long terminal repeat region was analyzed by strong stop cDNA synthesis and S1 nuclease mapping. The length of the RU5 region was determined and found to be 346 nucleotides long. The 5' long terminal repeat is 1,123 base pairs long and is bound by an 18-base-pair primer-binding site complementary to the 3' end of mammalian lysine-1,2-specific tRNA. Open reading frames for gag and pol genes were identified. Surprisingly, the HSRV gag protein does not contain the cysteine motif of the nucleic acid-binding proteins found in and typical of all other retroviral gag proteins; instead the HSRV gag gene encodes a strongly basic protein reminiscent of those of hepatitis B virus and retrotransposons. The carboxy-terminal part of the HSRV gag gene products encodes a protease domain. The pol gene overlaps the gag gene and is postulated to be synthesized as a gag/pol precursor via translational frameshifting analogous to that of Rous sarcoma virus, with 7 nucleotides immediately upstream of the termination codons of gag conserved between the two viral genomes. The HSRV pol gene is 2,730 nucleotides long, and its deduced protein sequence is readily subdivided into three well-conserved domains, the reverse transcriptase, the RNase H, and the integrase. Although the degree of homology of the HSRV reverse transcriptase domain is highest to that of murine leukemia virus, the HSRV genomic organization is more similar to that of human and simian immunodeficiency viruses. The data justify classifying the spumaretroviruses as a third subfamily of Retroviridae.
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PMID:Analysis of the primary structure of the long terminal repeat and the gag and pol genes of the human spumaretrovirus. 245 55


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