Conditional lethal mutations are very useful in genetic and biochemical analyses of complex processes such as DNA replication. Temperature sensitive (ts) mutations, which are one form of conditional lethal mutation, allow growth at one temperature (for example, 30oC) but not at a higher temperature (for example, 42oC).
A large number of temperature sensitive replication mutants have ben isolated from E. coli. These mutant bacteria are defective in DNA replication at 42oC, these mutants stop making DNA in one of two characteristic ways. The quick-stop’ mutants halt DNA synthesis immediately whereas the slow-stop’ mutants stop DNA synthesis only after many minutes.
Predict which of the following proteins, if temperature sensitive, would display a quick-stop phenotype and which would display a slow-stop phenotype.
In each case, explain your prediction:
DNA topoisomerase I
Single-strand binding protein
Cell-free extracts of the mutants show essentially the same pattern of replication as the intact cells. Extracts from quick-stop mutants halt DNA synthesis immediately at 42oC, whereas extracts from slow-stop mutants do not stop DNA synthesis for several minutes after a shift to 42oC. Suppose extracts from a temperature-sensitive DNA helicase mutant and temperature-sensitive DNA ligase mutant were mixed together at 42oC. Would you expect the mixture to exhibit a quick-stop phenotype, a slow-stop phenotype or a non-mutant phenotype?
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