Robert A. Weisberg

Prophage lambda at unusual chromosomal locations: I. Location of the secondary attachment sites and the properties of the lysogens

Abstract The integration, frequency of phage λ into a mutant host deleted for the normal prophage insertion site is reduced about 200-fold relative to integration into wild-type Escherichia coli. This residual integration, like normal integration, requires the int gene of the phage and occurs by a cross-over at the normal attachment site on the phage chromosome. Analysis of the resulting abnormal lysogens indicates that there are certain sites on the mutant bacterial chromosome which are preferentially utilized for prophage insertion. In addition, lysogens in which λ has inserted into or near a specific gene, thereby inactivating its function, may be obtained by the appropriate selection technique. When a bacterial gene has been inactivated by prophage integration, prophage excision can restore its function. Prophage excision from the abnormal sites is inefficient but, like excision from the normal site, it requires two phage genes: int and xis. In this respect the abnormal bacterial attachment sites resemble the normal bacterial attachment site rather than a phage or a hybrid attachment site. The abnormal lysogens are of value for deletion mapping of various portions of the E. coli chromosome and for the production of novel types of transducing phages.

T4 Endonuclease VII Cleaves Holliday Structures

T4 endonuclease VII cleaves Holliday structures in vitro by cutting two strands of the same polarity at or near the branch point. The two unbranched duplexes produced by cleavage each contain a strand break that can be sealed by DNA ligase. This suggests that the cut sites are at the same position in the nucleotide sequence in each strand. The joint action of endonuclease VII and DNA ligase can therefore resolve Holliday structures into genetically sensible products. These observations account for the role of endonuclease VII in the DNA metabolism of phage T4, and provide the first example of an enzyme that acts specifically on branch points in duplex DNA.

Site-specific Recombination in Phage Lambda

INTRODUCTION Cells lysogenic for λ carry a quiescent form of the viral chromosome called prophage. The prophage differs from the DNA of viral particles in two important ways: (1) The ends of the prophage and of the viral particle DNA are at different points in the nucleotide sequence, and (2) the prophage ends are covalently joined to host DNA. Campbell (1962) proposed that viral particle DNA is converted to prophage by the joining of the ends followed by insertion of the resulting circular molecule into the host DNA. Insertion occurs by reciprocal recombination at specific sites (attachment or att sites) in each chromosome (Fig. 1). This proposal has received extensive experimental support and, indeed, was generally accepted when the first edition of this book was written (see Gottesman and Weisberg 1971). A stable lysogen is formed when an infecting viral particle succeeds both in repressing lytic functions and in inserting its DNA. The inserted prophage is then replicated as part of the bacterial chromosome. In the rare event that repression is not followed by insertion (abortive lysogeny), the viral chromosome cannot replicate and so is lost by dilution as the host cell divides. Excision of the prophage from the chromosome is rare during normal bacterial growth but occurs readily following repressor inactivation. If the repressor is inactivated only briefly (abortive or transient derepression), prophage excision can occur without lytic growth. The excised DNA is then frequently lost as the cell divides (prophage curing). Intricate controls ensure that insertion occurs only...

Packaging of coliphage lambda DNA. II. The role of the gene D protein.

Abstract The gene D protein (p D ) of coliphage λ is normally an essential component of the virus capsid. It acts during packaging of concatemeric λ DNA into the phage prohead and is necessary for cutting the concatemers at the cohesive end site ( cos ). In this report we show that cos cutting and phage production occur without p D in λ deletion mutants whose DNA content is less than 82% that of λ wild type. D -independence appears to result directly from DNA loss rather than from inactivation (or activation) of a phage gene. (1) In cells mixedly infected with undeleted λ and a deletion mutant, particles of the deletion mutant alone are efficiently produced in the absence of p D ; and (2) D -independence cannot be attributed to loss of a specific segment of the phage genome. p D -deficient phage resemble p D -containing phage in head size and DNA ends; they differ in their extreme sensitivity to EDTA, greater density, and ability to accept p D . p D appears to act by stabilizing the head against disruption by overfilling with DNA rather than by changing the capacity of the head for DNA. This is shown by the observation that the amount of DNA packaged by a “headful” mechanism, normally in excess of the wild-type chromosome size, is not reduced in the absence of p D . In fact, p D is required for packaging headfuls of DNA. This implies that a mechanism exists for preventing the entry of excess DNA into the head during packaging of concatemers formed by deletion mutants, and we suggest that this is accomplished by binding of cos sites to the head. The above results show that p D is not an essential component of the nuclease that cuts λ concatemers at cos during packaging, and they imply that 82% of a wild-type chromosome length can enter the prohead in the absence of p D . Yet, p D is needed for the formation of cohesive ends after infection with undeleted phage. We propose two models to account for these observations. In the first, cos cutting is assumed to occur early during packaging. The absence of p D leads to release of packaged DNA and the loss of cohesive ends by end-joining. In the second, cos cutting is assumed to occur as a terminal event in packaging. p D promotes cos cutting indirectly through its effect on head stability. We favor the second model because it better explains the asymmetry observed in the packaging of the chromosomes of cos duplication mutants (Emmons, 1974).

Prophage lambda at unusual chromosomal locations: II. Mutations induced by bacteriophage lambda in Escherichia coli K12☆

An Escherichia coli strain deleted for the primary λ attachment site was lysogenized with λ at secondary sites. Some lysogens became mutants because of prophage insertion in the affected gene. Mutagenesis by phage λ is not random with respect to the gene affected: most mutants were pro, although certain other genes could be mutated at lower frequencies. In the case of several independent ilv and gal mutants, the sites of prophage insertion were in the same segment of the ilv region and galT gene respectively. The galT location may also be a preferred site for the insertion of DNAs other than prophage λ. Insertion of prophage λ within an operon can reduce the expression of operator-distal genes. A trpC λ insertion mutant expresses the operator-distal trpB function constitutively at a low level. This expression probably derives from a promoter located in the left arm of the prophage.

Role for DNA homology in site-specific recombination: The isolation and characterization of a site affinity mutant of coliphage λ*

Site-affinity (or saf) mutations change the specificity of prophage insertion. We have isolated a saf mutation of the bacteriophage lambda attachment site by inserting the phage chromosome into and then excising it from a secondary host attachment site. This causes reciprocal exchange of two seven base-pair segments (the overlap regions) that lie within the cores of the two sites. Since the two overlap regions differ from each other in nucleotide sequence, the recombinant sites are mutants. We have determined the effect of overlap region homology on recombination. We found that homology promotes integrative and excisive recombination. This suggests that the two overlap regions interact directly during recombination. The pattern of segregation of the saf mutation during site-specific recombination shows that it lies to the right of the point of genetic exchange about 95% of the time. This is a surprising result because lambda integrative recombination normally occurs by two staggered, reciprocal single-strand exchanges, one at each edge of the overlap region (Mizuuchi et al., 1981). Since saf lies within the overlap region, we might have expected that the point of genetic exchange would occur to the left of saf as often as to the right. We offer two models to account for this. (1) The mutation alters the location of one of the single-strand exchange points. (2) Efficient and strand-specific processing of mismatched base-pairs changes the expected segregation pattern.

Identifying Determinants of Recombination Specificity: Construction and Characterization of Chimeric Bacteriophage Integrases

Bacteriophage integrases are members of a family of structurally related enzymes that promote recombination between DNA molecules that carry specific sites. Phages lambda and HK022 encode closely related integrases that recognize different sets of sequences within the core regions of their respective attachment sites. To locate the amino acid residues that determine this difference in specificity, we isolated recombinant phages that produce chimeric integrases and measured the ability of these chimeras to promote recombination of lambda and HK022 sites in vivo. A chimera that is of lambda origin except for one HK022 residue at position 99 and 12 HK022 residues located between positions 279 and 329 had wild-type HK022 specificity and activity for both integrative and excisive recombination. Chimeras containing certain subsets of these 13 residues had incomplete specificity. The region around position 99 is not well-conserved in other members of the integrase family, but the 279-329 segment includes residues that are highly conserved and believed to be directly involved in catalysis. Many chimeras were inactive in recombining either HK022 or lambda sites. Selection for mutants that restored activity to these chimeras revealed sets of residues that are likely to interact with each other.

A genetic analysis of the att-int-xis region of coliphage lambda.

We have isolated and characterized several hundred mutants of bacteriophage λ that are defective in prophage excision. Almost all resemble classical int and xis mutants in complementation pattern and ability to integrate. One int mutant is exceptional in that it is defective in excision but proficient in integration. The mutant sites are located in a region that begins within 50 base-pairs to the right of the attachment site and extends about 1350 base-pairs rightward, int is approximately 1240 base-pairs and xis 110 base-pairs long.

Lambda integrase cleaves DNA in cis.

In the Int family of site‐specific recombinases, DNA cleavage is accomplished by nucleophilic attack on the activated scissile phosphodiester bond by a specific tyrosine residue. It has been proposed that this tyrosine is contributed by a protomer bound to a site other than the one being cleaved (‘trans’ cleavage). To test this hypothesis, the difference in DNA binding specificity between closely related integrases (Ints) from phages lambda and HK022 was exploited to direct wild type Ints and cleavage‐ or activation‐defective mutants to particular sites on bispecific substrates. Analysis of Int cleavage at individual sites strongly indicates that DNA cleavage is catalyzed by the Int bound to the cleaved site (‘cis’ cleavage). This conclusion contrasts with those from previous experiments with two members of the Int family, FLP and lambda Int, that supported the hypothesis of trans cleavage. We suggest explanations for this difference and discuss the implications of the surprising finding that Int‐family recombinases appear capable of both cis and trans mechanisms of DNA cleavage.

Enzymes and sites of genetic recombination: studies with gene 3 endonuclease of phage T7 and with site affinity mutants of phage lambda

The ability of bacteriophage T7 endonuclease I to cleave Holliday structures was invesigated. Holliday structures are an intermediate in genetic recombination, and their cleavage is essential for bateriophage growth. 37 references, 11 figures.