Franklin W. Stahl

The double-strand-break repair model for recombination

Gene conversion is the nonreciprocal transfer of information from one DNA duplex to another; in meiosis, it is frequently associated with crossing-over. We review the genetic properties of meiotic recombination and previous models of conversion and crossing-over. In these models, recombination is initiated by single-strand nicks, and heteroduplex DNA is generated. Gene conversion is explained by the repair of mismatches present in heteroduplex DNA. We propose a new mechanism for meiotic recombination, in which events are initiated by double-strand breaks that are enlarged to double-strand gaps. Gene conversion can then occur by the repair of a double-strand gap, and postmeiotic segregation can result from heteroduplex DNA formed at the boundaries of the gap-repair region. The repair of double-strand gaps is an efficient process in yeast, and is known to be associated with crossing-over. The genetic implications of the double-strand-break repair model are explored.

Radiation-sensitivity of bacteriophage containing 5-bromodeoxyuridine☆☆☆

Abstract Bacteriophage containing 5-bromodeoxyuridine have increased sensitivity to X-rays, ultraviolet light (2537 A), and light of longer wavelengths emanating from commercial fluorescent fixtures. The increase in sensitivity for the last case amounts to a factor of 75 for phage T2 u when fully substituted. A proffered hypothesis to account for the increased sensitivity is based on a comparative study of UV-resistant and UV-sensitive strains of T2.

Rec-mediated recombinational hot spot activity in bacteriophage lambda: III. Chi mutations are site-mutations stimulating Rec-mediated recombination☆☆☆

Lambda phage defective for Red and gam function make small plaques on rec+ bacteria. Mutants (called Chi) of λ arise which suppress the small-plaque phenotype. Chi mutations arise at at least four well-separated sites; one site is between gene L and att, one is between att and gam, one is in the cII gene, and one is near gene S. A phage strain carrying a Chi mutation at a given site has an extraordinarily high rate of Rec-mediated crossing-over near that site.

Chi sites in combination with RecA protein increase the survival of linear DNA in Escherichia coli by inactivating exoV activity of RecBCD nuclease.

In Escherichia coli, unprotected linear DNA is degraded by exoV activity of the RecBCD nuclease, a protein that plays a central role in the repair of double‐strand breaks. Specific short asymmetric sequences, called chi sites, are hotspots for RecBCD‐promoted recombination and are shown in vitro to attenuate exoV activity. To study RecBCD‐chi site interactions in vivo we used phage lambdas terminase to introduce a site‐specific double‐strand break at lambdas cos site inserted into a plasmid. We show that after terminase has cut cos in vivo, nucleases degrade linearized DNA only from the end that does not have a strong terminase binding site. Linearized cosmid DNA containing chi sites in the proper orientation to the unprotected end is degraded more slowly in rec+ E. coli than is chi‐less DNA. Increased survival of chi‐containing DNA is a result of partial inactivation of exoV activity and is dependent on RecA and SSB proteins. The linearization of chi‐containing DNA molecules leads to RecA‐dependent formation of branched structures which have been proposed as intermediates in the RecBCD pathway of double‐strand break repair.

A role for recombination in the production of “free-loader” lambda bacteriophage particles

Abstract Density-labeled crosses were performed with bacteriophage lambda under conditions which diminish DNA duplication. The production of viable phage containing fully conserved parental DNA was found to be dependent upon the action of the genetic recombination systems. The production of phage containing DNA with one newly synthesized chain was less dependent upon recombination. The production of phage with chromosomes both of whose chains were synthesized following infection show little, if any, dependence on recombination. One can speculate that some step in the maturation process of bacteriophage lambda is inseparable from the reduction of lambda DNA to the monomeric rods characteristic of lambda virions.

Improved in vitro packaging of coliphage lambda DNA: a one-strain system free from endogenous phage

In previous systems for in vitro packaging of lambda DNA, phages are produced from the packaging components as well as from added DNA. We have developed a new genetic strategy for in vitro packaging that bypasses this endogenous phage problem. Our system employs a single bacterial strain whose lambda prophage codes for all of the packaging proteins but is deleted for cos, the packaging origin. Crude extracts of the single lysogen: (i) are virtually free from endogenous phages, (ii) package added lambda DNA efficiently and (iii) are easy to prepare. Using the cos- in vitro packaging system we show that packaging of lambda linear monomers is a second-order reaction, but that packaging from concatemers prepared by annealing or ligation is first order. We conclude that in our cos- system, linear monomers are a poor substrate for in vitro packaging but that packaging from concatemers works well.

Double-strand breaks can initiate meiotic recombination in S. cerevisiae

We investigated the effects of double-strand breaks on meiotic recombination in yeast. A double-strand break was introduced at the MATa locus by sporulation of a MAT alpha inc/MATa diploid under inducing conditions for the HO-encoded endonuclease; 14% of the resulting tetrads had undergone 4 alpha:0a conversion. Conversion at MAT was associated with co-conversion of a closely linked marker and an increased recombination frequency for flanking markers. We also studied the sporulation products of a diploid heterozygous at the HIS4 locus for an insertion of a 100 bp fragment of MATa containing the HO endonuclease cut site. Under inducing conditions, a significant number of tetrads were formed that had undergone gene conversions in favor of the HIS4+ allele. Although double-strand breaks can initiate meiotic recombination in yeast, the data suggest that they do not normally do so.

Rec-mediated recombinational hot spot activity in bacteriophage λ

SummaryA Chi mutation in phage λ stimulates Rec-mediated crossing over more to one side of itself than to the other; stimulation, which is maximal near Chi, can occur at some distance from the Chi site as well. A gross heterology differentiating the two recombining parents does not interfere with the distant Chi-stimulated crossover whether the heterology is at the Chi site or between the Chi site and the distant interval in which recombination is monitored. These conclusions hold whether recombination is measured “genetically” in standard crosses or “physically” in density-labeled crosses conducted in the absence of DNA replication.

Properties of the DNA-delay mutants of bacteriophage T4☆

Abstract The intracellular events following infection with T4 DNA delay mutants have been examined in detail. The mutants fall into four complementation groups, rather than five as had been thought. Both the eclipses and latent periods of the mutants are extended in comparison to wild type. DNA synthesis was followed after infection by diphenylamine assay and by uptake of radioactive thymidine. DNA replication by the mutants appears to initiate at the normal time but at a reduced rate. Density transfer experiments support this conclusion. After 10–15 min of replication, the rate of DNA synthesis increases markedly. Net synthesis of late protein also is affected in these mutants since, in contrast to wild type, the rate of uptake of radioactive leucine declines at 15–20 min after infection. Appearance of phage lysozyme is delayed several minutes; appearance of g23 protein is delayed 5–10 minutes and the final rate of synthesis is reduced as much as 2-fold. Net RNA synthesis as measured by uptake of radioactive uracil appears normal, but phenotypic reversion experiments using 5-fluorouracil suggest that the synthesis of late messenger may be delayed in some of the mutants.

Orientation-dependent recombination hotspot activity in bacteriophage λ☆

Promoters of genetic exchange by the Escherichia coli Rec system, Chi elements, have been analyzed in λ phages carrying bacterial EcoRI restriction fragments. Some fragments confer Chi+ phenotype in one orientation and Chi− in the opposite orientation. The inactivity of Chi in one orientation explains why all active Chi elements in λ manifest a certain recombinational bias of the same sense. When these studies were undertaken, we rather expected to find two classes of Chi, one class which stimulated recombinant formation stronger to its left and one class stimulating recombinant formation more strongly to its right. The failure to find the second class is now understandable by supposing that the orientation of Chi which would have permitted it to act rightward is the orientation in which Chi has no activity at all. Several models are proposed for the orientation dependence of Chi activity.