DNA double-chain breaks in recombination of phage lambda and of yeast.
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Bacteriophage lambda↗
Biomedical subjects
D S Thaler
Publications and source records attributed to D S Thaler.
Double-chain-cut sites are recombination hotspots in the Red pathway of phage lambda.
The Red recombination pathway of phage lambda is shown to target recombination to double-chain ends of DNA. A double-chain cut, delivered in vivo to only one of two parents participating in a lambda lytic cross by a type II restriction endonuclease, increases the proportion of crossing over in the interval containing the cut compared with other intervals. The stimulating effect of a cut is evident whether replication is inhibited or permitted. Cut stimulation can move away from the initial cut-site, presumably by double-chain degradation. Movement of the stimulating effect of a cut is dependent on the Escherichia coli gene recA when the cross is carried out under conditions that inhibit phage replication. When replication is permitted, all aspects of cut-stimulated recombination are independent of recA. Evidence is presented to show that the reaction that is stimulated by cutting is often non-reciprocal at the molecular level.
Bacteriophage lambda↗
Evidence that the normal route of replication-allowed Red-mediated recombination involves double-chain ends.
Recombination mediated by the Red pathway of bacteriophage lambda is focused towards sites of double-chain cuts. Double-chain ends created either by type II restriction enzymes acting at unmodified recognition sites or by lambda's packaging enzyme, terminase, acting at cos are utilized in a manner similar to the double-chain break repair pathway of recombination in yeast. When lambda is allowed to recombine during replicative growth, spontaneous recombination is approximately evenly distributed along the chromosome. It has been proposed that replication-allowed recombination also is initiated by double-chain ends. In order to test this hypothesis we ask if the in vivo expression of the Mu gam protein is inhibitory to Red recombination. Mu gam has been shown in vitro to bind to linearized duplex DNA and to shield bound DNA from exonucleases. The expression of Mu gam is found to be inhibitory to Red recombination whether replication is blocked or allowed. As a control we ask if Mu gam inhibits Int-mediated recombination. It has been well documented that the Int pathway of recombination does not involve any double-chain breaks and, consistent with this, the Int pathway is not inhibited by Mu gam. We suggest that the in vivo expression of Mu gam or other similar activities may be a generally useful way to determine if those processes that respond to an artificially introduced double-chain cut normally involve double-chain ends.
Bacteriophage lambda↗
Tests of the double-strand-break repair model for red-mediated recombination of phage lambda and plasmid lambda dv.
The double-strand-break repair (DSBR) model was formulated to account for various aspects of yeast mitotic and meiotic recombination. In this study three features of the DSBR model are tested for Red-mediated recombination between phage lambda and lambda dv, a plasmid that is perfectly homologous to about 10% of lambda. The results support the applicability of the DSBR model to lambda's Red system: (1) Creating a double-strand-break (DSB) within the region of homology shared by phage and plasmid increases their genetic interaction by about 20-fold. A DSB outside the region of shared homology has no such effect. (2) Both patches, i.e., simple marker rescue, and splices, i.e., co-integration of the phage and plasmid, are stimulated by a DSB in the region of shared homology. (3) Co-integrants harbor a duplication of the region of shared homology. Among co-integrants that were formed by the creation of a DSB, there is a preferential loss of whichever allele was in cis to a utilized cut site. The DSBR model as originally formulated involves the isomerization and cleavage of Holliday junctions to resolve the canonical intermediate. We propose as an alternative mechanism that a topoisomerase can resolve the canonical DSBR intermediate.
Bacteriophage lambda↗
Hereditary stability and variation in evolution and development.
Evolution and development are both lineage processes but are often conceptualized as occurring by different and mutually exclusive mechanisms. It is conventionally asserted that evolution occurs via the random generation of diversity and the subsequent survival of those that pass selection. On the other hand, development is too often presented as proceeding via the unfolding of a deterministic program encoded in the DNA sequence. In biology, universal generalizations are rare and dogmas are often wrong for particular cases. Deterministic mechanisms contribute some of the new DNA sequences that subsequently become substrates for natural selection. Conversely, stochastic and selective mechanisms are intrinsic to development, and also to maintenance of the immune, and possibly, nervous systems. Cancer appears to be another process that straddles distinctions between evolutionary and developmental modes of hereditary change and stabilization. DNA sequence changes are an essential feature of many cancers, but there are also aspects of the disease similar to developmental lineage gone awry. The literature suggests that the cellular changes that give rise to cancer occur by mechanisms commonly associated with both evolutionary and developmental lineage pathways.
Animals↗