Читать книгу Snyder and Champness Molecular Genetics of Bacteria - Tina M. Henkin - Страница 68

Most of the genes for replication proteins have been found by isolating mutants defective in DNA replication, but not RNA or protein synthesis. Since a mutant cell that cannot replicate its DNA will die, any mutation (for definitions of mutants and mutations, see “Replication Errors” below and chapter 3) that inactivates a gene whose product is required for DNA replication will kill the cell. Therefore, for experimental purposes, only a type of mutant called a temperature-sensitive mutant can be usefully isolated with mutations in DNA replication genes. These are mutants in which the product of the gene is active at one temperature but inactive at another. The mutant cells can be propagated at the temperature at which the protein is active (the permissive temperature). However, shifting to the other (nonpermissive) temperature can test the effects of inactivating the protein. The molecular basis of temperature-sensitive mutants is discussed in more detail in chapter 3.

The immediate effect of a temperature shift on a mutant with a mutation in a DNA replication gene depends on whether the product of the gene is continuously required for replication at the replication forks or is involved only in the initiation of new rounds of replication. For example, if the mutation is in a gene for DNA polymerase III or in the gene for the DnaG primase, replication ceases immediately. However, if the temperature-sensitive mutation is in a gene whose product is required only for initiation of DNA replication, for example, the gene for DnaA or DnaC (see “Initiation of Chromosome Replication” below), the replication rate for the population will slowly decline. Unless the cells have been somehow synchronized in their cell cycles, each cell is at a different stage of replication, with some cells having just finished a round of replication and other cells having just begun a new round. Cells in which rounds of chromosome replication were under way at the time of the temperature shift will complete their replication cycle but will not start a new round. Therefore, the rate of replication decreases until the rounds of replication in all the cells are completed.