Genetic deletions between directly repeated sequences in bacteriophage T7

Abstract

DNA sequence analysis of genetic deletions in bacteriophage T7 has shown that these chromosomal rearrangements frequently occur between directly repeated DNA sequences. To study this type of spontaneous deletion in more quantitative detail synthetic fragments of DNA, made by hybridizing two complementary oligonucleotides, were introduced into the non-essential T7 gene 1.3 which codes for T7 DNA ligase. This insert blocked synthesis of functional ligase and made the phage that carried an insert unable to form plaques on a host strain deficient in bacterial ligase. The sequence of the insert was designed so that after it is put into the T7 genome the insert is bracketed by direct repeats. Perfect deletion of the insert between the directly repeated sequences results in a wild-type phage. It was found that these deletion events are highly sensitive to the length of the direct repeats at their ends. In the case of 5 bp direct repeats excision from the genome occurred at a frequency of less than 10(-10), while this value for an almost identical insert bracketed by 10 bp direct repeats was approximately 10(-6). The deletion events were independent of a host recA mutation.

Visualization of Repair of Double-Strand Breaks in the Bacteriophage T7 Genome without Normal DNA Replication

“…Extracts were prepared with T7 ⌬A 3 genome. Because there are no repeats at its ends, the insert will not delete from the genome at detectable frequency and cannot grow on a lig-7(Ts) host (34,35). Genes 1.3, 1.4, 1.5, 1.6, and 1.7 are non-essential (48), so T7 with this insert and deletion can grow normally on a wild-type E. coli host.…”

“…Amber mutants used in the extract preparations included am29 in gene 3 (endonuclease I), am28 in gene 5 (DNA polymerase), and am147 in gene 6 (5Ј 3 3Ј exonuclease). The ⌬A mutation is a deletion from the promoter of gene 1.3 (T7 ligase) to gene 1.5 (35). (The functions of the nonessential genes 1.4 and 1.5 have not been determined.)…”

“…In some experiments, inserts of DNA were placed in an XhoI site that had been engineered at position 6663 in the T7 ligase gene (gene 1.3). The construction of this phage, designated T7X, has been described previously (35). The construction and sequences of the specific T7 inserts used in this study have been described in detail previously (21).…”

Deletion mutagenesis independent of recombination in bacteriophage T7

“…Phage which underwent deletion between the direct repeats had the same frequency of recombination between the left and right flanking markers as was found in controls in which no deletion events took place. These data argue against intermolecular recombination between direct repeats as a major factor in deletion in T7-infected E. coli.Deletions often arise between directly repeated DNA sequences (1,7,11,18,33,38,42,44,48). Short regions of homology probably play a crucial role in the mechanism(s) responsible for this type of spontaneous deletion.…”

“…The small size of the repeats distinguishes illegitimate recombination events (such as those associated with transposition, slip mispairing, and deletion) from homologous recombination, which is dependent upon extensive regions of homology (2,6,16). However, some studies show that deletions are RecA dependent (1, 3, 4), while others show no RecA dependence (6,7,11,14,15,33,53 (22,32,37,43). The only known host protein important to T7 DNA replication is thioredoxin, which associates with the phage gene 5 product and confers extensive processivity upon the DNA polymerase (37).…”