Experimental approaches for the study of DNA replication
Three different approaches have been used for the study of DNA replication in prokaryotes : (i) Isolation of mutants. Replication defective conditional mutants have been selected, which are, temperature sensitive dnats (non-conditional mutants, inhibiting DNA synthesis are usually lethal and are therefore not useful for a study of DNA replication.).DNA synthesis in these mutants stops, when shifted to non-permissive temperature (e.g. 45°C). These can be either the replication initiation/termination mutants (also called 'slow stop mutants') which do not stop DNA synthesis immediately after shifting to non-permissive temperature (e.g. dnaA, dnaB and dnaC), or may be replication extension/elongation mutants (also called 'quick stop mutants'), which stop DNA synthesis immediately after shifting (e.g. dnaB, dnaC, dnaE, dnaG, dnaN and dnaX). Thus the proteins synthesized by the former class must be involved in initiation/termination and those synthesized by the latter must be involved in elongation. In former class, DNA synthesis does not stop immediately, because DNA synthesis continues for one complete round on replication units where DNA synthesis was already initiated. Some of the viable mutants, which are not temperature sensitive, but are defective in DNA replication, have also been found useful, (ii) Use of inhibitors. Sensitivity of the initiation of DNA synthesis to rifampicin suggested that RNA synthesis is involved in the initiation of DNA replication. 'Novobiocin' and 'nalidixic acid' inhibited elongation by inhibiting 'DNA gyrase', which functions as a replication swivel, (iii) Isolation of proteins. Several proteins, which have a role in DNA synthesis, have been identified, by either their ability to complement the defective mutants or by their requirement for in vitro replication of plasmid or phage DNA. Genes for several of these proteins have now been isolated and cloned.