In vitro repair of double-strand breaks accompanied by recombination in bacteriophage T7 DNA

Abstract

A double-strand break in a bacteriophage T7 genome significantly reduced the ability of that DNA to produce viable phage when the DNA was incubated in an in vitro DNA replication and packaging system. When a homologous piece of T7 DNA (either a restriction fragment or T7 DNA cloned into a plasmid) that was by itself unable to form a complete phage was included in the reaction, the break was repaired to the extent that many more viable phage were produced. Moreover, repair could be completed even when a gap of about 900 nucleotides was put in the genome by two nearby restriction cuts. The repair was accompanied by acquisition of a genetic marker that was present only on the restriction fragment or on the T7 DNA cloned into a plasmid. These data are interpreted in light of the double-strand gap repair mode of recombination.A popular model for repair of a double-strand break, based upon the recombination of the broken molecule with an intact portion of a homologous region of duplex DNA, is supported by considerable experimental evidence (23,(36)(37)(38)(39). In this model, the double-strand break is first widened to form a gap so that each strand of both pieces of the broken DNA can establish base pairing with complementary strands on the intact homolog. Repairlike DNA synthesis with the unbroken donor molecule as template would fill the gap. The recipient DNA molecule which had suffered a strand break thereby acquires genetic information present on the donor molecule. There is also considerable evidence that phage T4 recombination is stimulated at the ends of DNA molecules (17). This paper describes repair of a double-strand break in the highly recombinogenic (34, 35) bacteriophage T7. T7 recombination is independent of the host RecA recombinase (21), and T7 is known to inactivate exonuclease V, the product of the Escherichia coli recBCD genes (43). It has been suggested that T7 single-strand-binding protein may play a central role in promoting the annealing of complementary single strands of phage DNA (28). T7 endonuclease I (gene 3), helicase-primase (gene 4), DNA polymerase (gene 5), and exonuclease (gene 6) play roles in T7 recombination (5,7,11,12,22,26,40,41). The precise molecular mechanisms responsible for T7 recombination are poorly understood. Therefore, possible involvement of strand breaks in this phage's recombination mechanism(s) remains an open question.Other workers have reported in vitro recombination of T7 DNA (6,9,(25)(26)(27)(28)(29)42 MATERIALS AND METHODSBacteria and phage. Bacteria used in this study were the wild-type, suppressor-free E. coli strain W3110, E. coli TB-1 [ara A(lac-proAB) rpsL hsdR D80 lacZM15], the suppressorfree Shigella sonnei strain ShD2 371-48, and S. sonnei Sh3-18, which contains an amber suppressor. Both S. sonnei strains and the ss-("suicide in Shigella") T7 strain were gifts from R. Hausmann. Wild-type T7 and T7 with amber mutation am29 in gene 3, am20 in gene 4, am28 in gene 5, or am147 in gene 6 were from W. Studier. Our previous studies had shown that a single A-...

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

“…The yield of viable phage reflects the number of intact genomes and, therefore, the efficiency of double-strand break repair. Double-strand breaks are repaired efficiently in this system, and repair of the breaks is often accompanied by acquisition of genetic information from other DNA molecules present in the same reactions (25). When a double-strand break occurs between a pair of direct repeats, the break can increase the frequency of deletion of the region between the repeats by 2 or more orders of magnitude (13,55).…”

“…A connection between double-strand breaks and recombination (both homologous and illegitimate) has been well established in a number of biological systems, including yeasts, bacteria, and bacteriophages (9,12,32,44,46,53,54). Our laboratory has been examining the repair of double-strand breaks by using an in vitro system based on extracts made from Escherichia coli infected with bacteriophage T7 (13,21,25,55). In this system, DNA replication closely mimics the in vivo replication of T7 DNA (4, 28).…”

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

“…Recombination (2) and DNA repair (14,15) are extremely efficient in T7-infected cells even in the absence of the host RecA pathway (16). gp6 plays a critical role in recombination as evidenced by the absence of DNA exchange when E. coli is infected with bacteriophage T7 deficient in gp6; using density labeling, no hybrid T7 molecules are observed (17).…”

Gene 4 helicase of bacteriophage T7 mediates strand transfer through pyrimidine dimers, mismatches, and nonhomologous regions

“…We have proposed, on the basis of in vitro studies, that the efficient recombination observed in phageinfected cells is mediated by the T7 gene 2.5 protein and the T7 gene 4 helicase (2). Furthermore, recombinational repair of dsDNA breaks in T7 phage-infected cells is very efficient (15,16). The studies presented here show that the helicasemediated strand transfer reaction has the capability to play a major role in postreplication recombinational repair.…”

“…Recombination (14) and DNA repair (15,16) in E. coli cells infected with bacteriophage T7 are extremely efficient, and recombination is not impaired by the absence of the host RecA pathway (17). Consequently, we believe that the T7 gene 4 helicase-mediated strand exchange reaction provides the major pathway for recombination in E. coli cells infected with bacteriophage T7 (2).…”