Biomedical subjects

K D Lunnen

Publications and source records attributed to K D Lunnen.

9 recordsLinked to original sources

PubMed2001

Engineering a nicking endonuclease N.AlwI by domain swapping.

Changing enzymatic function through genetic engineering still presents a challenge to molecular biologists. Here we present an example in which changing the oligomerization state of an enzyme changes its function. Type IIs restriction endonucleases such as AlwI usually fold into two separate domains: a DNA-binding domain and a catalytic/dimerization domain. We have swapped the putative dimerization domain of AlwI with a nonfunctional dimerization domain from a nicking enzyme, N.BstNBI. The resulting chimeric enzyme, N.AlwI, no longer forms a dimer. Interestingly, the monomeric N.AlwI still recognizes the same sequence as AlwI but only cleaves the DNA strand containing the sequence 5'-GGATC-3' (top strand). In contrast, the wild-type AlwI exists as a dimer in solution and cleaves two DNA strands; the top strand is cleaved by an enzyme binding to that sequence, and its complementary bottom strand is cleaved by the second enzyme dimerized with the first enzyme. N.AlwI is unable to form a dimer and therefore nicks DNA as a monomer. In addition, the engineered nicking enzyme is at least as active as the wild-type AlwI and is thus a useful enzyme. To our knowledge, this is the first report of creating a nicking enzyme by domain swapping.

Amino Acid Sequence↗

PubMed1997

Overexpression of BsoBI restriction endonuclease in E. coli, purification of the recombinant BsoBI, and identification of catalytic residues of BsoBI by random mutagenesis.

BsoBI is a type II restriction enzyme found in Bacillus stearothermophilus JN209 that recognizes the symmetric sequence 5'-CYCGRG-3' (Y=C or T; R=A or G) and cleaves between the first and second base to generate a four-base 5' extension. The cloning and sequencing of BsoBI restriction-modification system has been described by Ruan et al. [Mol. Gen. Genet. 252 (1996) 695-699]. Here we report the overexpression of BsoBI restriction endonuclease gene in E. coli by insertion of the endonuclease gene into an expression vector pRRS. The recombinant BsoBI was purified to homogeneity and its N-terminus sequence was determined. It has the same N-terminal aa sequence as the native enzyme. The constitutive expression of BsoBI from pRRS is lethal to E. coli in the absence of the cognate methylase. The bsoBIR gene was mutagenized with either hydroxylamine or by error-prone polymerase chain reaction in vitro and transferred into E. coli via plasmid vectors in the absence of the cognate methylase. Surviving transformants were selected that carry BsoBI variants which lost endonuclease activity. DNA sequencing of the mutant alleles revealed that G123, D124, D212, D246, E252 and H253 are important residues for enzymatic activity. An electrophoretic mobility shift assay was used to identify binding-proficient and cleavage-deficient variants. Seven variants I95M&D124Y, G123R, D212N, K207R&D212V, D246N, D246G and E252K can still bind DNA despite the loss of cleavage activity. Thus, residues D124, D212, D246 and E252 may be located near or within the catalytic center, and are likely involved in metal ion binding.

Amino Acid Sequence↗

PubMed1996

Cloning and sequence comparison of AvaI and BsoBI restriction-modification systems.

AvaI and BsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5'CYCGRG3' and cleave between the first C and second Y to generate a four-base 5' extension. The AvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned into Escherichia coli by the methylase selection method. The BsoBI restriction endonuclease gene (bsoBIR) and part of the BsoBI methylase gene (bsoBIM) were cloned by the "endo-blue" method (SOS induction assay), and the remainder of bsoBIM was cloned by inverse PCR. The nucleotide sequences of the two restriction-modification (RM) systems were determined. Comparisons of the predicted amino acid sequences indicated that AvaI and BsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity. Although the two systems show similarity in protein sequence, their gene organization differs. The avaIM gene precedes avaIR in the AvaI RM system, while the bsoBI R gene is located upstream of bsoBI M in the BsoBI RM system. Both AvaI and BsoBI methylases contain motifs conserved among the N4 cytosine methylases.

Amino Acid Sequence↗

PubMed1989

Characterization and cloning of MwoI (GCN7GC), a new type-II restriction-modification system from Methanobacterium wolfei.

R.MwoI, a type-II restriction enzyme with the new specificity 5'-GCN7GC-3', was found in extracts of the thermophilic archaebacterium, Methanobacterium wolfei. R.MwoI cleaves duplex DNA producing fragments with 3-nt, 3'-terminal extensions, thus: GCN5/N2GC. The genes coding for the MwoI restriction and modification enzymes were cloned into Escherichia coli on the plasmid vector pBR322. The clones synthesize a low level of R.MwoI endonuclease. The plasmids display incomplete MwoI-specific modification, suggesting that the clones synthesize a low level of the M.MwoI methyltransferase, too.

Cloning, Molecular↗

PubMed1988

Cloning type-II restriction and modification genes.

We have cloned into Escherichia coli the genes for 38 type-II bacterial modification methyltransferases. The clones were isolated by selecting in vitro for protectively modified recombinants. Most of the clones modify their DNA fully but a substantial number modify only partially. In approximately one-half of the clones, the genes for the corresponding endonucleases are also present. Some of these clones restrict infecting phages and others do not. Clones carrying endonuclease genes but lacking methyltransferase genes have been found, in several instances, to be viable.

Bacterial Proteins↗

PubMed1988

Cloning and sequencing the HinfI restriction and modification genes.

The HinfI restriction and modification genes were cloned on a 3.9-kb PstI fragment inserted into the PstI site of plasmid pBR322. Both genes are confined to an internal 2.3-kb BclI-AvaI subfragment. This subfragment was sequenced. Two large open reading frames (ORF's) are present. ORF1 codes for the methylase [predicted 359 amino acids (aa)] and ORF2 codes for the endonuclease (predicted 262 or 272 aa).

Amino Acid Sequence↗

PubMed1984

Complete concordance between glucose-6-phosphate dehydrogenase activity and hypomethylation of 3' CpG clusters: implications for X chromosome dosage compensation.

To explore the molecular basis of X chromosome inactivation, we have examined the human locus for glucose-6-phosphate dehydro-genase (G6PD) in various human tissues. Studies of DNA from males and females and from somatic cell hybrids with active or inactive X chromosomes, show that two remarkably dense clusters of CpG dinucleotides in the 3' coding sequences are hypomethylated in active G6PD genes but extensively methylated in inactive ones. Reacquisition of G6PD activity, either spontaneous or induced by 5-azacytidine, is accompanied by demethylation of both clusters; however, the clusters remain methylated in reactivants that express HPRT but not G6PD. Our observations implicate these 3' CpG clusters in the transcription of G6PD and in maintenance of dosage compensation for X linked housekeeping genes.

Alleles↗

PubMed1984

Methylation of the hypoxanthine phosphoribosyltransferase locus on the human X chromosome: implications for X-chromosome inactivation.

To explore the role of DNA methylation in maintaining dosage compensation of X chromosome-linked genes and in regulating the transcriptional activity of "housekeeping" genes, we characterized DNA methylation of active, inactive, and derepressed alleles at the locus for hypoxanthine phosphoribosyltransferase (HPRT) on the human X chromosome. The methylation of Hpa II and Hha I sites in HPRT alleles on the active X chromosome was the same in all tissues. The consensus pattern includes hypomethylation of 5' clustered sites and extensive methylation of the 3' sequence. The striking feature of methylation of inactive X-chromosome alleles is nonuniformity and less extensive hypomethylation of the 5' cluster. Analysis of HPRT alleles reactivated in response to 5-azacytidine showed at least partial restoration of the consensus pattern. These observations indicate that methylation of housekeeping genes on the X chromosome is the same as that of autosomal ones and that the overall pattern and methylation of multiple sites within a cluster may cooperate to facilitate transcription. Furthermore, the fidelity of methylation of the active allele and the extensive drift in methylation of the inactive allele suggest that mechanisms involved in X-chromosome dosage compensation may be directed at the active rather than inactive X chromosome.

Alleles↗

PubMedDate not supplied

Sequence comparison of the EcoHK31I and EaeI restriction-modification systems suggests an intergenic transfer of genetic material.

The genes coding for the EcoHK31I and EaeI restriction-modification (R-M) systems from Escherichia coli strain HK31 and Enterobacter aerogenes, respectively, have been cloned and sequenced. Both ENases recognize and cleave Y/GGCCR leaving 4 nucleotide 5'-protruding ends, while the MTases modify the internal cytosine. The systems were isolated on a 2.3kb AseI fragment for EcoHK31I, and a 4.6 kb HindIII fragment for EaeI. The R and M genes of both systems converge and overlap by 14 nucleotides. Previously, we found that M.EcoHK31I consisted of two subunits, (alpha and beta), with the beta subunit being translated from an alternative open reading frame within the gene encoding the alpha subunit. Sequence comparison between the EcoHK31I and EaeI systems reveals striking similarity. The eaeIM gene also encodes alpha and beta polypeptides of 309 and 176 amino acids which share 96% and 97% identity, respectively, with those of ecoHK31IM. ecoHK31IR and eaeIR encode proteins of 318 and 315 aa, respectively, which share 92% identity but are otherwise unique in the GenBank database. The EaeI and the EcoHK31I R-M systems were found to be flanked by genes coding for integrases. It is possible that these integrases have facilitated the transfer of this system among different bacterial species.

Amino Acid Sequence↗