We have isolated a genomic DNA fragment from HeLa cells containing the promoter region and the first two exons of the human gene encoding DNA topoisomerase I (hTOPI). Transcription of hTOPl mRNA initiates at multiple sites which are clustered 247 nucleotides and 210 nucleotides upstream of the translation-initiation site of the protein coding region. The nucleotide sequence of the region preceding the transcription-initiation sites is G/C rich and contains sequence motifs which are known binding sites of the transcription factors Octl (octameric transcription factor l), Spl and AP2 (activator protein 2). Furthermore, one CAMP-responsive element is present 50 nucleotides upstream of the transcription-initiation site nearest the 5' end. Neither TATA nor CAAT boxes were found in the promoter region of the hTOPl gene. A 918-bp fragment containing the sequence elements described above drives the transient expression of a chloramphenicol acetyl transferase (CAT) gene sequence in transfected HeLa and 293 cells. In addition we analyzed a 10-kb fragment containing the promoter and exons 1 and 2 for regions of DNase I hypersensitivity. We detected one prominent DNase-I-hypersensitive region in the promoter close to the putative transcription-factor-binding sites and several weaker regions in intron 2.Human type I DNA topoisomerase (hTOP1) is a monomeric protein with an apparent molecular mass of 100 kDa. In vitro, the purified enzyme catalyzes the relaxation of positive and negative superhelical turns in DNA molecules by successive cycles of single-strand breakage and rejoining of the phosphodiester bonds of the DNA backbone (reviewed in [l -31). About 106TOP1 molecules/nucleus are present in mammalian cells. They are translated from a 4.2-kb poly(A)+ mRNA which is encoded by the single hTOPl gene located on chromosome 20q11.2-13.1 [4, 51.In the eukaryotic cell the double-stranded DNA is organized as a nucleoprotein complex and thereby topologically restrained. Transient changes in the torsional tension of this DNA molecule can be generated by exogeneous stimuli or induced endogeneously by the processes of transcription or DNA replication. These changes result in an alteration of twist and writhe in topologically restrained DNA molecules. Active topoisomerases recognize these topological changes and catalyze the relaxation of DNA by changing the linking number (reviewed in [6]).There is overwhelming evidence that TOP1 is one of the two major enzymes controlling the torsional tension of chromatin in eukaryotic cells. It has been shown that TOP1 (together with topoisomerase 11) acts as swivelase in DNA replication and removes the superhelical tension which would otherwise accumulate as a consequence of replication fork
Different subfragments of a cDNA coding for DNA topoisomerase I were used as probes to determine the chromosomal localization of topoisomerase I sequences in human cells. Southern blotting of restricted DNA from a panel of rodent-human somatic cell hybrids revealed the localization of the complete gene on chromosome 20 and the presence of two truncated topoisomerase I pseudogene sequences on chromosomes 1 and 22. In situ chromosome hybridization experiments confirmed these results showing the location of the complete gene on band q11.2-13.1 of chromosome 20, and the location of the pseudogene sequences on band q23-24 of chromosome 1 and q11.2-13.1 of chromosome 22.
From rat brain extracts, two carnosine-degrading enzymes have been identified and partially purified by ion-exchange chromatography, hydrophobic interaction chromatography on phenyl-Sepharose CL4B and gel filtration. These enzymes exhibit distinct differences in their chemical characteristics and substrate specificities.One enzyme, designated carnosinase, preferentially hydrolyzes carnosine and exhibits a low K, value (0.02 mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine-degrading enzyme hydrolyzes PAla-Arg considerably faster than carnosine and, therefore, has been tentatively designated PAla-Arg hydrolase. This enzyme exhibits high K , values with carnosine ( K , = 25 mM) and PAla-Arg ( K , = 2 mM). Homocarnosine and y-aminobutyryl-arginine are not degraded by PAlaArg hydrolase.Neither enzyme is inhibited by agents reactive on activated hydroxyl groups, such as diisopropyl fluorophosphate, and also not by a variety of peptidase inhibitors of microbial origin or from other sources. Carnosinase is also not inhibited by bestatin but PAla-Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50 = 50 nM). While carnosinase is strongly inhibited by thiolreducing agents such as dithioerythritol and 2-mercaptoethanol, PAla-Arg hydrolase is stabilized and activated by these substances. Both enzymes are strongly inhibited by metal-chelating agents. Carnosinase, however, is not dependent on exogeneously added metal ions and is strongly inhibited by MnZ+ as well as by heavy metal ions. In contrast, BAla-Arg hydrolase requires MnZ + ions for full enzymatic activity. Based on these differences, selective incubation conditions could be evaluated in order to determine specifically both enzyme activities in crude tissue extracts. In rat, both enzymes are present in all tissues tested, except skeletal muscles. but considerable differences in their relative distribution among different tissues are also observed.As early as 1900, carnosine had been isolated from Liebig's meat extract [l] and subsequently identified as P-alanylhistidine [2, 31. Since then, various w-aminoacyl amino acids such as anserine (j-alanyl-1 -methylhistidine), homocarnosine (y-aminobutyryl-histidine), /I-alanylornithine, P-alanyl-lysine etc. have been isolated from excitable tissues, brain and muscle (for reviews see [4] and [5]).Biochemical studies with cell-free tissue extracts strongly indicated that all w-aminoacyl amino acids are synthesized by only one enzyme, carnosine synthetase [L-histidine : p-alanine ligase (AMP forming), EC 6.3.2.111, which is present in brain and muscle [6,7]. In agreement with these results, we recently demonstrated that carnosine and related dipeptides are actively synthesized by glial cells in culture [8]. In order to understand the mechanisms regulating the metabolism of w-aminoacyl amino acids, we were interested in studying the degradation of these peptides by brain enzymes. Here we describe the id...
We have isolated from a Lambda-gt 11 library a human cDNA clone with one open reading frame of about 2400 bases. A stretch of about 350 amino acids in the deduced amino acid sequence is up to 40 percent identical with parts of the known amino acid sequences of E. coli and yeast glutaminyl (Gln)-tRNA synthetase. The isolated cDNA sequence corresponds to an internal section of a 5500 bases long mRNA that codes for a 170 kDa polypeptide associated with Gln-tRNA synthetase. Thus, the human enzyme is about three times larger than the E. coli and two times larger than the yeast Gln-tRNA synthetase. The three enzymes share an evolutionarily conserved core but differ in amino acid sequences linked to the N-terminal and C-terminal side of the core.
We examined the promoter of the human type-I-DNA topoisomerase gene (hTOP1) for regions protected against DNase I digestion by nuclear proteins from HeLa or from adenovirus-transformed 293 cells. We identified ten protected DNA sequences within 580 bp of DNA upstream of the transcriptional-start sites and one additional site, which is located between the two clusters of transcriptional-start sites. Several of these protein-binding sites have significant similarities to recognition sequences of known transcription factors including factors Spl, octamer transcription factor, CAMP-responsive-element-binding protein (CREB/ATF), NF-KB and members of the Myc-related family of basichelix-loop-helidleucine-zipper proteins. Other protein-binding sites show less or no similarities to known consensus sequences. We investigated the physiological significance of these protein-binding sites using a set of deletion and nucleotide-exchange mutants. We conclude that the expression of the hTOPl gene is regulated by a complex network of negatively and positively acting transcription factors.Topoisomerases are enzymes present in all nucleated cells. They catalyse the relaxation of torsional tension in DNA and participate in all processes requiring the separation of complementary strands in topologically fixed doublestranded DNA. Their participation in DNA replication (Avemann et al., 1988;Holm et al., 1989;Yang et al., 1987; for reviews see Richter and Knippers, 1989;Zhang et al., 1990), transcription (Brill and Sternglanz, 1988;Liu and Wang, 1987;Ostrander et al., 1990;Stewart et al., 1990) and DNA recombination (Bullock et al., 1985;Rose et al., 1990; Wallis et al., 1989; and for a recent review Wang et a]., 1990) has been demonstrated.Eukaryotic type-I DNA topoisomerases resolve positive and negative superhelical turns by introducing transient single-stranded nicks with the simultaneous covalent binding of the enzyme to one DNA end. The intact strand is then passed through the open strand before the nick is rejoined using the energy stored in the diester bond between a protein tyrosine and the DNA 3' phosphate end.Human type-I DNA topoisomerase (hTOPl), a monomeric enzyme of 91 kDa, is encoded by a single gene, lo-
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