Abstract
The T7 chromosome is a double-stranded linear DNA molecule flanked by direct terminal repeats or so-called terminal redundancies. Late in infection bacteriophage T7 DNA accumulates in the form of concatemers, molecules that are comprised of T7 chromosomes joined in a head to tail arrangement through shared terminal redundancies. To elucidate the molecular mechanisms of concatemer processing, we have developed extracts that process concatemeric DNA. The in vitro system consists of an extract of phage T7-infected cells that provides all T7 gene products and minimal levels of endogenous concatemeric DNA. Processing is analyzed using a linear 32P-labeled substrate containing the concatemeric joint. T7 gene products required for in vitro processing can be divided into two groups; one group is essential for concatemer processing, and the other is required for the production of full length left-hand ends. The products of genes 8 (prohead protein), 9 (scaffolding protein), and 19 (DNA maturation) along with gene 18 protein are essential, indicating that capsids are required for processing. In extracts lacking one or more of the products of genes 2 (Escherichia coli RNA polymerase inhibitor), 5 (DNA polymerase), and 6 (exonuclease), full length right-hand ends are produced. However, the left-hand ends produced are truncated, lacking at least 160 base pairs, the length of the terminal redundancy. Gene 3 endonuclease, required for concatemer processing in vivo, is not required in this system. Both the full length left- and right-hand ends produced by the processing reaction are protected from DNase I digestion, suggesting that processing of the concatemeric joint substrate is accompanied by packaging.
Highlights
The T7 chromosome is a double-stranded linearDNA synthesis.Leadingstrandsynthesisextendsthe3’end of molecule flanked by directterminalrepeats or so- nascent DNA to the 5‘ end of the template
To elucidatethe molecular the chromosome, and the 3‘ ends of template mechanisms of concatemer processing, we have developed extracts that process concatemeric DNA
The in vitro system consists of an extracotf phage T7-infected cells that provides all T7 gene products and minimal levels of endogenous concatemeric DNA
Summary
Extensive genetic and.physica evidence has shown that concatemer processing in T7 and other phages occurs after concatemeric DNA has bound to capsids and cliossely coupled to DNA packaging in vivo (Serweret al., 1983; Feiss and restrictive to the growth of T7(1.2-) phage (Saito and Richardson, 1981). The extract shouldprovide all gene products conceivably important for packaging, and processing, of concatemeric DNA. Packaging endogenous concatemeric DNA whose presence would compete with exogenous substrate Using these guidelines, a packaging extract was prepared by combining a whole cell lysate of T7,,,-infected cells, which provides capsidsandmaturationproteins,with acleared. Upon gentle lysis and centrifugation, the concatemeric DNA is found inthe pellet fraction, leavinga supernatantalmostfree of DNA (datanot shown) This cleared lysate isadded to lysed T7,,,-infected cells which lack a. To. test this proposal, packaging extracts derived from a combination of T74,s- and T7,*-infected E. coli B, which contain endogenous gene 18 protein, were supplemented with an ex-.
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