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Biomedical subjects

J Jiricny

Publications and source records attributed to J Jiricny.

At least 73 records · Page 4Linked to original sources
PubMed1994

Efficient removal of uracil from G.U mispairs by the mismatch-specific thymine DNA glycosylase from HeLa cells.

The uracil DNA glycosylases (EC 3.2.2.3) characterized to date remove uracil from DNA irrespective of whether it is base paired with adenine or mispaired with guanine in double-stranded substrates or whether it is found in single-stranded DNA. We report here the characterization of uracil glycosylase activity that can remove the base solely from a mispair with guanine. It does not recognize uracil either in A.U pairs or in single-stranded substrates. The enzyme, a 55-kDa polypeptide, was previously characterized as a mismatch-specific thymine DNA glycosylase and was thought to be responsible solely for the correction (to G.C) of G.T mispairs that arise as a result of spontaneous hydrolytic deamination of 5-methylcytosine to thymine. Given the broader substrate specificity of the enzyme (in addition to uracil and thymine, the protein can also remove 5-bromouracil from mispairs with guanine), we propose that its biological role in vivo may also include the correction of a subset of G.U mispairs inefficiently removed by the more abundant ubiquitous uracil glycosylases, such as those arising from cytosine deamination in G+C-rich regions of the genome.

Base Composition↗
PubMed1994

Colon cancer and DNA repair: have mismatches met their match?

Recently, both a common inherited human cancer, hereditary non-polyposis colorectal carcinoma (HNPCC), and certain sporadic human cancers have been shown to have frequent alterations in microsatellite sequences. These were ascribed to a defect in the correction of errors of replication; indeed, the cell line H6, which is derived from an HNPCC tumour, has been shown to be deficient in mismatch repair. The HNPCC locus on chromosome 2p has been linked to a gene hMSH2, which encodes a 100 kDa mismatch-binding protein that has an extensive degree of sequence conservation from bacteria through yeast to humans. But could a malfunction of this protein alone cause cancer?

Amino Acid Sequence↗
PubMed1994

Localization of a 34-amino-acid segment implicated in dimerization of the herpes simplex virus type 1 ICP4 polypeptide by a dimerization trap.

The herpes simplex virus type 1 immediate-early protein ICP4 plays an essential role in the regulation of the expression of all viral genes. It is the major trans activator of early and late genes and also has a negative regulatory effect on immediate-early gene transcription. ICP4 is a sequence-specific DNA-binding protein and has always been purified in a dimeric form. The part of the protein that consists of the entire highly conserved region 2 and of the distal portion of region 1 retains the ability to specifically associate with DNA and to form homodimers in solution. In an attempt to map the dimerization domain of ICP4, we used a dimerization trap assay, in which we screened deletion fragments of this 217-amino-acid stretch for sequences that could confer dimerization properties on a heterologous cellular transcription factor (LFB1), which binds to its cognate DNA sequence only as a dimer. The analysis of these chimeric proteins expressed in vitro ultimately identified a stretch of 34 amino acids (343 to 376) that could still confer DNA-binding activity on the LFB1 reporter protein and thus apparently contained the ICP4 dimerization motif. Consistent with this result, a truncated ICP4 protein containing amino acids 343 to 490, in spite of the complete loss of DNA-binding activity, appeared to retain the capacity to form a heterodimer with a longer ICP4 peptide after coexpression in an in vitro translation system.

Amino Acid Sequence↗
PubMed1993

The purification of a mismatch-specific thymine-DNA glycosylase from HeLa cells.

G/T mispairs that arise in the DNA of higher eukaryotes as a result of spontaneous hydrolytic deamination of 5-methylcytosine to thymine must be corrected to G/C pairs. We describe here the purification to apparent homogeneity of the enzyme that initiates this repair process by excising the mispaired thymine from the hetero-duplex to generate an apyrimidinic site. The enzymatic activity could be attributed to a 55-kDa polypeptide, which was purified from extracts of HeLa cells by a combination of conventional and DNA-affinity chromatography. The enzyme is a mismatch-specific thymine-DNA N-glycosylase, capable of hydrolyzing the carbon-nitrogen bond between the sugar-phosphate backbone of the DNA and a mispaired thymine. In addition to the G/T, the enzyme can remove thymine also from C/T and T/T mispairs in the order G/T >> C/T > T/T. It has no detectable endonucleolytic activity on apyrimidinic sites and does not catalyze the removal of thymine from A/T pairs or from single-stranded DNA.

Base Sequence↗
PubMed1992

The purification of a human mismatch-binding protein and identification of its associated ATPase and helicase activities.

A mismatch-binding protein has been purified an estimated 4500-fold from HeLa nuclear extracts using four different chromatographic steps. Two polypeptides of apparent molecular weight of 160,000 and 100,000 were present in the final affinity-purified fraction as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Partial proteolytic clipping of the protein-DNA complexes visualized after UV treatment indicated that the 100-kDa polypeptide is most likely a degradation product of the 160-kDa polypeptide. UV cross-linking experiments have shown that both these polypeptides bind specifically to oligonucleotide duplexes containing G/T mismatches. Direct DNA binding studies and band-shift competition assays showed that although the mismatch-binding protein binds with highest affinity to oligonucleotides containing G/T mismatches, it is also capable of binding to oligonucleotides containing other mispairs. The purified protein has an associated Mg(2+)-dependent ATPase activity, which is markedly enhanced in the presence of single-stranded DNA. A helicase capable of unwinding a 34-mer oligonucleotide, annealed to a complementary sequence in single-stranded M13, also copurified with the mismatch-binding protein. This reaction occurs in an ATP- and magnesium-dependent manner.

Adenosine Triphosphatases↗
PubMed1992

The mode of cauliflower mosaic virus propagation in the plant allows rapid amplification of viable mutant strains.

We inoculated the leaves of turnip plants (Brassica campestris spp. rapa cv. Just Right) with two cauliflower mosaic viruses (CaMVs) with different small mutations in a dispensable region of the viral genome, and followed the spread of the virus infection through the plant. Surprisingly, analysis of viral DNA in single primary chlorotic lesions revealed the presence of both mutants. In contrast, the secondary chlorotic lesions and systemically infected leaves contained virus molecules of either one or the other type only. Infection of plants with different ratios of the two reporter viruses showed that this ratio is not conserved during systemic virus spread. Infection with CaMV DNA in the form of heteroduplexes containing a single mismatched base pair, in which each strand carried a distinct diagnostic marker, provided us with evidence that the mismatch was subjected to a repair process in the host plant.

Base Sequence↗
PubMed1991

Effect of uracil situated in the vicinity of a mispair on the directionality of mismatch correction in Escherichia coli.

We wanted to establish whether strand breaks and gaps, arising during the removal of uracil from newly-synthesized DNA, can be utilized as strand discrimination signals by the methyl-directed mismatch repair system of Escherichia coli. For this purpose, we constructed a series of M13 heteroduplexes that contained a single uracil residue situated either upstream or downstream from a G/T or an A/C mispair. Transfections of these constructs into E. coli strains, either proficient of deficient in mismatch or uracil repair, allowed us to follow the fate of these mispairs in vivo. Our data show that the intermediates of uracil repair cannot substitute for the strand-discrimination signals generated by the MutH protein, which is thought to initiate the methyl-directed mismatch repair process by nicking the unmethylated strand of a newly-synthesized DNA duplex at d(GATC) sites. However, processing of uracil residues situated upstream from the mispair was shown to reduce the yield of the progeny phage arising from the uracil-containing strand, presumably as a result of co-repair of the base analogue and the mispair.

Amino Acid Sequence↗
PubMed1990

The reverse transcriptase gene of cauliflower mosaic virus is translated separately from the capsid gene.

Cauliflower mosaic virus (CaMV) possesses start codons at the beginning of its reverse transcriptase (RT) gene (ORF V) suggesting that, unlike in retroviruses and retrotransposons, it is translated independently from the capsid gene (ORF IV). To test this hypothesis a mutational analysis of the CaMV ORF IV/V overlapping region was performed. Mutants in which both ORFs are separated by stop codons in all three reading frames are viable and stable, while mutations affecting the first two AUG codons of ORF V are either lethal or unstable, giving rise to true and second site reversions. Mutants in which the AUG codons were replaced by ACG or AAG reverted only slowly and ACG could direct the synthesis of small amounts of reporter enzyme in transfected plant protoplasts, showing that this codon can act in plant cells as a weak start codon. CaMV has apparently developed a strategy for translation of the RT gene different from that in retroviruses and retrotransposons, but similar to that of hepadnaviruses, another group of pararetroviruses. The separate translation of the RT gene as a common feature of pararetroviruses might reflect the difference in their life cycle in comparison with retroviruses.

Base Sequence↗
PubMed1990

Open reading frame VIII is not required for viability of cauliflower mosaic virus.

Open reading frame (ORF) VIII of cauliflower mosaic virus (CaMV) was analyzed by site-directed mutagenesis in order to investigate its potential function for the viral life cycle. Removal of either the start or the stop codon of ORF VIII, as well as interruption of ORF VIII by a new stop codon, did not affect infectivity. Unlike certain ORF VII mutants all three ORF VIII mutants are stable. Hence the ORF VIII product is not essential and ORF VIII mutations do not have deleterious polar effects on the expression of the downstream ORF V, which codes for the viral protease/reverse transcriptase.

Codon↗
PubMed1990

Mismatch-specific thymine DNA glycosylase and DNA polymerase beta mediate the correction of G.T mispairs in nuclear extracts from human cells.

To avoid the mutagenic effect of spontaneous hydrolytic deamination of 5-methylcytosine, G.T mispairs, arising in DNA as a result of this process, should always be corrected to G.C pairs. We describe here the identification of a DNA glycosylase activity present in nuclear extracts from HeLa cells, which removes the mispaired thymine to generate an apyrimidinic (AP) site opposite the guanine. We further show, using a specific antibody and inhibitors, that the single nucleotide gap, created upon processing of the AP site, is filled in by DNA polymerase beta. This finding substantiates the proposed role of this enzyme in short-patch DNA repair.

Aphidicolin↗
PubMed1989

Cytosine methylation in CTF and Sp1 recognition sites of an HSV tk promoter: effects on transcription in vivo and on factor binding in vitro.

We methylated specific cytosine residues within or immediately around the CTF and Sp1 binding sites of the Herpes simplex virus thymidine kinase promoter. The efficiency of transcription in vivo was reduced at least 50-fold compared with transcription from the unmethylated promoter. However, methylation within the CTF recognition site had no effect on the affinity of CTF for this site in vitro. Methylation of the Sp1 site resulted in only a small decrease in the affinity of this factor for its recognition site. In vivo studies showed that the same gene inserted in different vector DNAs was regulated differently by methylation in the promoter. These results show that cytosine methylation can inhibit transcription by a mechanism other than directly blocking the binding of transcription factors.

Animals↗
PubMed1989

Purification and characterization of a protein from HeLa cells that binds with high affinity to the estrogen response element, GGTCAGCGTGACC.

A non-histone protein, NHP1, that binds with high affinity to the estrogen response element (ERE), GGTCAGCGTGACC, has been purified approximately 45,000-fold from HeLa cells by a combination of chromatography on Sephacryl S-300, heparin-Sepharose, Mono Q (FPLC), and sequence-specific oligonucleotide-Sepharose. The native protein has a molecular weight of 170,000 and is composed of two polypeptides of 85 and 75 kDa. The two polypeptides are different as judged by peptide mapping, and only the 85-kDa polypeptide can be cross-linked to the bromodeoxyuridine-substituted synthetic ERE by UV irradiation. The native protein binds to the ERE with an apparent KD of 1 x 10(-11) M and has a pI of 5. The contact points of the protein with individual bases of the ERE have been determined by using partially depurinated and depyrimidinated synthetic oligonucleotides. The strongest contact points of NHP1 with the ERE are 5'AGCG3' in the center of the palindrome and differ from those of the estrogen receptor. NHP1 appears to produce specific nicks around the central CpGs of the ERE, thereby suggesting that it may play a role in active demethylation of mCpGs.

Base Sequence↗
PubMed1989

In vitro correction of G.T mispairs to G.C pairs in nuclear extracts from human cells.

In differentiated cells, only a specific subset of genes is expressed. Recently, several genes have been shown to be transcriptionally inactivated by methylation of cytosine residues, mainly within their promoter sequences. Spontaneous hydrolytic deamination of 5-methylcytosine to thymine, which has been estimated to generate up to 12 G.T mismatched base pairs in the human genome per day, could have a deleterious effect on the expression of such genes. We recently reported that mammalian cells possess a specific repair pathway, which counteracts the mutagenic effects of this deamination by correcting G.T mismatches almost exclusively to G.C pairs. We show here that, in nuclear extracts from HeLa cells, this repair is mediated by excision of the aberrant thymidine monophosphate residue, followed by gap-filling to generate a G.C pair. We also provide preliminary evidence that the initial step of this process involves a DNA glycosylase.

Base Sequence↗
PubMed1989

Repair of base-base mismatches in simian and human cells.

Mismatched heteroduplexes arise as intermediates of several dissimilar genetic processes. The outcome of these genetic events will therefore be influenced by the efficiency and specificity of mismatch repair. We have studied the correction of base-base mispairs in simian and human fibroblasts by transfecting the cells with derivatives of SV40 DNA, each harboring a single mispair in a defined orientation. Analysis of plaques revealed that correction efficiencies for homomispairs followed the pattern G.G greater than C.C greater than or equal to A.A greater than T.T. Repair bias was influenced by flanking sequences. Correction efficiences for heteromispairs followed the pattern of G.T greater than A.C greater than C.T greater than A.G and repair favored the retention of G + C by a substantial margin. This repair specificity could lead to a gene conversion bias favoring the accumulation of G + C in sequences subject to high levels of recombination or unequal exchange.

Animals↗
PubMed1989

Different patterns of G/T and A/C mismatch repair in simian cells correspond to the specificity of a mismatch binding protein isolated from simian and HeLa cells.

The two transition mispairs G/T and A/C are corrected with different efficiencies and specificities in CV-1 African green monkey kidney cells. G/T mispairs are corrected with 99% efficiency and almost always in favor of guanine, while A/C mispairs exhibit a 31% uncorrected sector and are otherwise randomly corrected. The higher correction efficiency and bias for the G/T mispair can be correlated with the substrate specificity of a protein, found in HeLa and CV-1 cell extracts, that binds selectively to oligonucleotide duplexes containing G/T mispairs. No binding can be detected to duplexes containing other mispairs, or to homoduplexes.

Adenine↗