Biomedical subjects

M P MacWilliams

Publications and source records attributed to M P MacWilliams.

6 recordsLinked to original sources

PubMed1999

DNA translocation blockage, a general mechanism of cleavage site selection by type I restriction enzymes.

Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non-specific cleavage site. We have examined several potential blocks to DNA translocation, such as positive supercoiling or a Holliday junction, for their ability to trigger DNA cleavage by type I restriction enzymes. Introduction of positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzyme's ability to select cleavage sites randomly throughout the DNA molecule. Thus, positive supercoiling does not prevent DNA translocation. EcoR124II endonuclease cleaved DNA at Holliday junctions present on both linear and negatively supercoiled substrates. The latter substrate was cleaved by a single enzyme molecule at two sites, one on either side of the junction, consistent with a bi-directional translocation model. Linear DNA molecules with two recognition sites for endonucleases from different type I families were cut between the sites when both enzymes were added simultaneously but not when a single enzyme was added. We propose that type I restriction enzymes can track along a DNA substrate irrespective of its topology and cleave DNA at any barrier that is able to halt the translocation process.

DNA, Bacterial↗

PubMed1996

DNA cleavage by the type IC restriction-modification enzyme EcoR124II.

Type I restriction-modification systems bind to non-palindromic, bipartite recognition sequences. Although these enzymes methylate specific adenine residues within their recognition sequences, they cut DNA at sites up to several thousand base-pairs away. We have investigated the mechanism of how EcoR124II, a type IC restriction-modification system, selects the cleavage site. Restriction studies with different DNA constructs revealed that circular DNA requires only one non-methylated recognition sequence to be cut, whereas linear DNA needs at least two such sites. Cleavage of linear DNA is independent of site orientation. Further investigations of the linear substrates revealed a mechanism whereby the double-strand break is introduced between two recognition sequences. We propose a model for the selection of restriction sites by type I enzymes where two EcoR124II complexes bind to two recognition sequences. Lack of methylation at a site stimulates the enzyme to translocate DNA on both sides of the recognition sequence. Thus the two complexes approach each other and, at the point where they meet, they interact to introduce a double-strand break in the DNA.

Base Sequence↗

PubMed1996

Generation of new DNA binding specificity by truncation of the type IC EcoDXXI hsdS gene.

The hsdS subunit of a type IC restriction-modification enzyme is responsible for the enzyme's DNA binding specificity. Type I recognition sites are characterized by two defined half-sites separated by a non-specific spacer of defined length. The hsdS subunit contains two independent DNA binding domains, each targeted towards one DNA half-site. We have shown previously that the 5' half of hsdS can code for a functional substitute of the full-length hsdS. Here we demonstrate that the 3' half of the gene, when fused to the appropriate transcriptional and translational start signals, also codes for a peptide which imparts DNA binding specificity to the enzyme. About half the natural hsdS size, the mutant peptide contains a single DNA recognition domain flanked by one copy of each internal repeat found in the full-length hsdS. Deletion of either repeat sequence results in loss of activity. Like the 5' hsdS mutant, the 3' mutant recognizes an interrupted palindrome, GAAYN(5)RTTC, suggesting that two truncated subunits participate in DNA recognition. Co-expression of the 5' hsdS mutant and the 3' hsdS mutant along with hsdM regenerates the wild-type methylation specificity. Thus, there is a free assortment of subunits in the cell.

Adenine↗

PubMed1996

Genetic analysis of the bacteriophage lambda attL nucleoprotein complex.

Site-specific recombination in bacteriophage lambda involves interactions among proteins required for integration and excision of DNA molecules. We have analyzed the elements required to form an in vivo nucleoprotein complex of integrase (Int) and integration host factor (IHF). Interaction of Int with the core (the site of strand exchange) is stabilized by the flanking arm region of attL. IHF, in addition to Int, is required for efficient Int-core binding. We used the in vivo attL binding assay to characterize several Int variants for their abilities to form stable attL complexes. Substitution of Int active site tyrosine 342 by phenylalanine had no effect on the ability of the protein to form attL complexes. Three other amino acids that are completely conserved in the integrase family of recombinases (arginine 212, histidine 308, and arginine 311) were separately substituted by glutamine, leucine, and histidine, respectively. In each case, the mutant protein was altered in its ability to form attL complexes while retaining its ability to bind to the lambda arm-type sites. We propose that, in addition to their role in catalysis, this triad of amino acids helps the Int protein to interact with the lambda core sites.

Bacterial Proteins↗

PubMed1993

Direct genetic selection for a specific RNA-protein interaction.

The decision between lytic and lysogenic development of temperate DNA bacteriophages is determined largely by transcriptional regulation through DNA-binding proteins. To determine whether a heterologous RNA-binding activity could control the developmental fate of a DNA bacteriophage, a derivative of P22 was constructed in which the chosen developmental pathway is regulated by an RNA-binding molecule interacting with its RNA target site located in a phage mRNA. In the example presented, lysogenic development of the phage relies upon R17 coat protein expression in the susceptible host cell and the availability of a suitable coat protein binding site encoded by the phage genome. Through the analysis of phage mutants that are able to grow lytically in susceptible cells that express the coat protein, additional insights were obtained regarding the specific interaction of the R17 coat protein with its RNA binding site. This study also suggests a novel and extremely sensitive strategy for selecting RNA-binding activities in vivo.

Bacteriophage P22↗

PubMed1991

Genetic analysis of Escherichia coli integration host factor interactions with its bacteriophage lambda H' recognition site.

The bacteriophage P22-based challenge phage system was used to study the binding of integration host factor (IHF) to its H' recognition site in the attP region of bacteriophage lambda. We constructed challenge phages that carried H' inserts in both orientations within the P22 Pant promoter, which is required for antirepressor synthesis. We found that IHF repressed expression of Pant from either challenge phage when expressed from an inducible Ptac promoter on a plasmid vector. Mutants containing changes in the H' inserts that decrease or eliminate IHF binding were isolated by selecting challenge phages that could synthesize antirepressor in the presence of IHF. Sequence analysis of 31 mutants showed that most changes were base pair substitutions within the H' insert. Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IHF consensus binding site; mutants were isolated that contained substitutions at six of the nine base pairs of the consensus site. Other mutants contained changes at base pairs between the two subdeterminants of the H' site, at positions that are not specified in the consensus sequence, and in the dA + dT-rich region that flanks the consensus region of the site. Taken together, these results show that single-base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt IHF binding to the mutated site.

Bacterial Proteins↗