An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.

An in vitro system based on extracts of Escherichia coli infected with bacteriophage T7 is able to repair double-strand breaks in a T7 genome with efficiencies of 20% or more. To achieve this high repair efficiency it is necessary that the reaction mixtures contain molecules of donor DNA that bracket the double-strand break. Gaps as long as 1,600 nucleotides are repaired almost as efficiently as simple double-strand breaks. DNA synthesis was measured while repair was taking place. It was found that the amount of DNA synthesis associated with repair of a double-strand break was below the level of detection possible with this system. Furthermore, repair efficiencies were the same with or without normal levels of T7 DNA polymerase. However, the repair required the 533 exonuclease encoded by T7 gene 6. The high efficiency of DNA repair allowed visualization of the repaired product after in vitro repair, thereby assuring that the repair took place in vitro rather than during an in vivo growth step after packaging.Double-strand breaks in DNA confront the cell with potentially disastrous consequences, in the form of permanent loss of genetic information. Double-strand breaks can result from DNA-damaging agents (7), aberrant interactions between topoisomerases and DNA (11, 43), or from collapsed replication forks (2,19). To counteract the deleterious effects of double-strand breaks, most organisms maintain elaborate repair mechanisms directed against these lesions (3, 7). Recombination with undamaged portions of homologous genomes offers an economical scheme for rescue of partial genomes formed by double-strand breaks. 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). Moreover, the in vitro system is able to carry out at least some steps of homologous recombination (22,23,27,38,39). To study double-strand break repair, breaks are experimentally introduced with a restriction endonuclease at a predetermined site in the T7 genome. The broken genomes are then incubated in the in vitro system before the DNA is recovered and packaged into infective T7 phage. 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).Althou...

This study represents the largest series of patients undergoing office-based photoangiolytic laser treatment specifically for Reinke's edema. Our data suggest that this is a safe and effective modality to treat dysphonia associated with Reinke's edema, although patient intolerance of the procedure may represent a barrier.

SummaryAn in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to monitor repair of double-strand breaks in the T7 genome. The efficiency of double-strand break repair was markedly increased by DNA molecules (`donor' DNA) consisting of a 2.1 kb DNA fragment, generated by PCR, that had ends extending < 1 kb on either side of the break site. Repair proceeded with greater than 10% efficiency even when T7 DNA replication was inhibited. When the donor DNA molecules were labelled with 32 P, repaired genomes incorporated label only near the site of the double-strand break. When repair was carried out with unlabelled donor DNA and [ 32 P]-dCTP provided as precursor for DNA synthesis the small amount of incorporated label was distributed randomly throughout the entire T7 genome. Repair was performed using donor DNA that had adjacent BamHI and PstI sites. When the BamHI site was methylated and the PstI site was left unmethylated, the repaired genomes were sensitive to PstI but not to BamHI endonuclease, showing that the methyl groups at the BamHI recognition site had not been replaced by new DNA synthesis during repair of the double-strand break. These observations are most consistent with a model for double-strand break repair in which the break is widened to a small gap, which is subsequently repaired by physical incorporation of a patch of donor DNA into the gap.

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