Volume 3

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    from a primer; how a suitable primer sequence is provided is not known and we can only speculate on whether it might involve an initiation event at the same origin that is responsible for semiconservative replication of the duplex. Enzyme Activities Involved in Recombination Several types of enzyme activity must be necessary for recombination. The initial pairing reaction may require an activity capable of melting DNA—this might be provided by a protein analogous to the gene-32 protein of phage T4, which can preferentially maintain A-T rich regions of DNA in a denatured state (see Chapter 6). A nickase 1 activity able to make single strand breaks in the recipient duplex must be required at an early stage. Nuclease activities must be essential, although at present it is not possible to specify the precise roles that they take; however, their actions must be concerned with degrading the sequences that are replaced in the recipient genome. Of course, a single enzyme may have more than one of these activities. The model on the left of Figure 2.33, for example, illustrates a sequence of events in which a nick is made and the adjacent sequence of the recipient strand is then degraded; this could be achieved by the action of a nickase followed by an exonuclease but an alternative is to invoke a single enzyme with both endonuclease and exonuclease activi ties. Finally repair synthesis and ligase activities are necessary to achieve covalent linkage between the donor and recipient material. Isolation of mutants defective in recombination has identified several loci whose products presumably comprise recombination enzymes; but it is not yet possible to correlate any enzyme activities with specific stages in recombination. The ree' mutants of E.coli were first isolated by Clark and Margulies ( 1965) as variants of an F~ strain that were no longer able to produce haploid recombinants after conjugation with an Hfr strain; that is, these mutants cannot integrate the donor single strand into the recipient genome (see Clowes and Moody, 1966). Cells with any one of these mutations are defective not only in recombination but also display in creased sensitivity to both ultraviolet and X-irradiation. This suggests that the enzymes of this system may undertake repair of DNA damaged by irradiation as well as participating in a pathway for recombination. Since repair involves the removal of DNA sequences that are mispaired or represent structural distortions, such as the covalently linked thymine dimers generated by ultraviolet irradiation, it is not surprising that the mechanisms involved in repair and recombination may overlap. Repair of damaged DNA in E.coli is mediated by two principal systems: the uvr system undertakes excision-repair, that is to say that one strand of dam