Two proteins encoded by bacteriophage T7, the gene 2.5 single‐stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single‐stranded regions. The formation of a joint molecule consisting of circular, single‐stranded M13 DNA, annealed to homologous linear, duplex DNA having 3′‐ or 5′‐single‐stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5′ to 3′ direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5′‐triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein.
An in vitro system based on extracts of Escherichia coli infected with bacteriophage T7 was used to study genetic deletions between directly repeated sequences. The frequency of deletion was highest under conditions in which the DNA was actively replicating. Deletion frequency increased markedly with the length of the direct repeat both in vitro and in vivo. When a T7 gene was interrupted by 93 bp of nonsense sequence flanked by 20-bp direct repeats, the region between the repeats was deleted in about 1 out of every 1,600 genomes during each round of replication. Very similar values were found for deletion frequency in vivo and in vitro. The deletion frequency was essentially unaffected by a recA mutation in the host. When a double-strand break was placed between the repeats, repair of this strand break was often accompanied by the deletion of the DNA between the direct repeats, suggesting that break rejoining could contribute to deletion during in vitro DNA replication.Genetic deletions often occur between directly repeated sequences of DNA (1, 2, 5, 8). The presence of homologous sequences from 6 to 20 bp long at the ends of deleted regions brings to mind mechanisms that cause deletion by annealing complementary regions from direct repeats on opposite ends of the deleted sequence. Various models have been proposed to account for deletion mechanisms (2,5,8,11,18,24,31). Most of these can be classified into mechanisms that involve a form of copy choice during DNA synthesis (16,17,34,39,40), recombination between the direct repeats (2,3,8,10,19,30,33), or incorrect rejoining of DNA molecules which are broken as part of normal DNA metabolic activities (4,8,11,18,31,32,38). Rearrangements caused by switching of subunits between gyrase molecules resident at each of the direct repeats can be included in this latter category (13). The presence of nearby ends on DNA molecules undergoing deletion has been suggested as a common feature among these putative mechanisms (9). Achieving a clear understanding of deletion mechanisms has been difficult because of the complexity of biological events involved and the relatively infrequent occurrence of deletion. Nonetheless, the important roles of deletion in human inherited diseases, evolution, and biotechnology, as well as the potential insight into DNA replication and recombination mechanisms that may be reached by gaining a better understanding of how deletions occur, provide strong incentives for studying deletion mechanisms. In this study, we tested whether double-strand ends can stimulate deletion between direct repeats.We have been investigating deletions between direct repeats in bacteriophage T7 (14,25,26,28,29). The relative simplicity of its DNA replication process (27, 36) and the abundant biochemical and genetic information regarding T7 that has already been amassed (7,27,35,36) make T7 an especially interesting system with which to study deletions. We have adapted an in vitro DNA replication-packaging system that can
The frequency of genetic deletion between directly repeated DNA sequences in bacteriophage T7 was measured as a function of the length of the direct repeat. The non-essential ligase gene (gene 1.3) of bacteriophage T7 was interrupted with pieces of synthetic DNA bracketed by direct repeats of various lengths. Deletion of these 76 bp long inserts was too low to be measured when the direct repeats were less than 6 bp long. However, the frequency of deletion of inserts with longer direct repeats increased exponentially as the length of the repeats increased from 8 to 20 bp. When inverted repeats (palindromes) were designed in the midst of the insert there was essentially no increase in deletion frequency between 10 bp direct repeats. But, the same palindromic sequences increased the deletion frequency between 5 bp direct repeats by at least two orders of magnitude. Thus, in this system homology at the endpoints is a more important determinant of deletion frequency than is the presence of palindromes between the direct repeats.
In bacteriophage T7 the gene 2.5 singlestranded DNA-binding protein and the gene 4 helicase together promote the annealing of homologous regions of two DNA partners to form a joint molecule and subsequent strand transfer. In this reaction T7 gene 2.5 protein is essential for joint molecule formation, but is not required for T7 gene 4 protein-mediated strand transfer. T7 gene 4 helicase alone is able to mediate strand transfer, provided that a joint molecule is available. The present paper shows that, in addition, strand transfer proceeds at a normal rate even when both DNA partners contain ultraviolet-induced pyrimidine dimers (0.6 dimer per 100 nt). An insert of a relatively long (842-nt) segment of nonhomologous DNA in the single-stranded DNA partner has no effect on strand transfer, whereas its presence in the double-stranded partner prevents strand transfer. A short insert (37 nt) can be tolerated in either partner. Thus, DNA helicase is able to participate in recombinational DNA repair through its role in strand exchange, providing a pathway distinct from nucleotide excision repair.Recent studies have shown that the gene 41 DNA helicase encoded by bacteriophage T4 (1) and the gene 4 helicase encoded by bacteriophage T7 (2) mediate homologous DNA strand transfer within homologous DNA molecules. The rate and polarity of strand transfer are in accord with the properties of these enzymes in catalyzing strand displacement at a replication fork. A convenient DNA substrate for dissecting the strand transfer reaction is a joint DNA molecule formed between a single-stranded circle and a homologous linear duplex DNA molecule with a short 3Ј-single-stranded tail (Fig.
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