Biomedical subjects

M J Engler

Publications and source records attributed to M J Engler.

30 records · Page 2Linked to original sources

PubMed1983

Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase.

The DNA polymerase induced after infection of Escherichia coli by bacteriophage T7 can exist in two forms. One distinguishing property of Form I, the elimination of nicks in double-stranded DNA templates, strongly suggests that this form of the polymerase catalyzes limited DNA synthesis at nicks, resulting in displacement of the downstream strand. In this paper, we document this reaction by a detailed characterization of the DNA product. DNA synthesis on circular, duplex DNA templates containing a single site-specific nick results in circular molecules bearing duplex branches. Analysis of newly synthesized DNA excised from the product shows that the majority of the branches are less than 500 base pairs in length and that they arise from a limited number of sites. The branches have fully base-paired termini but are attached by two noncomplementary DNA strands that have a combined length of less than 30 nucleotides. The product molecules are topologically constrained as a result of the duplex branch. DNA sequence analysis has provided an unequivocal structure of one such product molecule. We conclude that strand displacement synthesis catalyzed by Form I of T7 DNA polymerase is terminated by a template-switching reaction. We propose two distinct models for template-switching that we call primer relocation and rotational strand exchange. Strand displacement synthesis catalyzed by Form I of T7 DNA polymerase effectively converts T7 DNA circles that are held together by hydrogen bonds in their 160-nucleotide-long terminal redundancy to T7-length linear molecules. We suggest that strand displacement synthesis catalyzed by T7 DNA polymerase is essential in vivo to the processing of a T7 DNA concatemer to mature T7 genomes.

Base Sequence↗

PubMed1983

Two forms of the DNA polymerase of bacteriophage T7.

The DNA polymerase induced by bacteriophage T7 can be isolated in two different forms. The distinguishing properties are: 1) the specific activities of the associated 3' to 5' single- and double-stranded DNA exonuclease activities, 2) the ability to catalyze DNA synthesis and strand displacement at nicks, and 3) the degree of stimulation of DNA synthesis on nicked, duplex DNAs by the gene 4 protein of phage T7. Form I is obtained when purification is carried out in the absence of EDTA while Form II is obtained if all purification steps are carried out in the presence of 0.1 mM EDTA. Form I has low levels of both exonuclease activities, less than 5% of those of Form II. Form I can initiate DNA synthesis at nicks leading to strand displacement, a consequence of which is its ability to be stimulated manyfold by the helicase activity of gene 4 protein on nicked, duplex templates. On the other hand, Form II cannot initiate synthesis at nicks even in the presence of gene 4 protein. In keeping with its higher exonuclease activities, Form II of T7 DNA polymerase has higher turnover of nucleotides activity (5-fold higher than Form I) and exhibits greater fidelity of nucleotide incorporation, as indicated by the rate of incorporation of 2-aminopurine deoxynucleoside monophosphate. Both forms of T7 DNA polymerase exhibit higher fidelity of nucleotide incorporation than bacteriophage T4 DNA polymerase. In the absence of EDTA or in the presence of FeSO4 or CaCl2, Form II irreversibly converts to Form I. The physical difference between the two forms is not known. No difference in molecular weight can be detected between the corresponding subunits of each form of T7 DNA polymerase as measured by gel electrophoresis in the presence of sodium dodecyl sulfate.

DNA Replication↗

PubMed1983

Bacteriophage T7 DNA replication. Synthesis of lagging strands in a reconstituted system using purified proteins.

Replication of the lagging strand of bacteriophage T7 DNA occurs in a discontinuous fashion that requires RNA-primed DNA synthesis, the removal of the RNA primers, the replacement of the ribonucleotides with deoxyribonucleotides, and the covalent joining of adjacent DNA fragments. We have examined each of these steps as well as the whole process through the use of model substrates and partial reactions using purified proteins. Tetraribonucleotides (pppACCC or pppACCA), synthesized by the T7 gene 4 protein on single-stranded DNA, are used as primers by T7 DNA polymerase to yield RNA-terminated DNA fragments. The removal of the RNA primers is catalyzed by the 5' to 3' hydrolytic activities of either Escherichia coli DNA polymerase I or the T7 gene 6 exonuclease. The products of hydrolysis are pppApC, ATP, and nucleoside 5'-monophosphates or ATP and nucleoside 5'-monophosphates, respectively. The requirement for DNA synthesis to fill the gap between adjacent DNA fragments can be fulfilled by Form II of T7 DNA polymerase but not by Form I. DNA synthesis catalyzed by Form II of T7 DNA polymerase eliminates gaps to create a substrate for DNA ligase whereas strand displacement synthesis catalyzed by Form I creates an aberrant structure that cannot be joined. Either the host or phage DNA ligase can effect the final covalent joining. All steps in the replication of a lagging strand have been coupled in a model system that catalyzes the formation of covalently closed, circular, double-stranded DNA molecules using single-stranded viral DNA as template. A combination of four bacteriophage proteins, gene 4 protein, Form II of T7 DNA polymerase, gene 6 exonuclease, and DNA ligase, can accomplish this overall reaction.

DNA Replication↗

PubMed1983