[43] Purification of bacteriophage T4 DNA replication proteins

Publisher Summary This chapter describes purification procedures for most of the T4 replication proteins. The bacteriophage T4 DNA replication system is a relatively simple system of ten T4 encoded proteins that together catalyze rapid and highly accurate copying of the two strands of a replication fork in vitro . The genes for most of the T4 replication proteins were first identified in studies of conditionally lethal phage mutants. These proteins were initially purified from T4 infected Escherichia coli using either complementation assays, which measured their ability to stimulate DNA synthesis by a crude extract of cells infected with a replication-defective T4 mutant, or functional assays of their ability to catalyze or stimulate specific replication reactions. The T4 proteins required for leading and lagging strand synthesis in vitro are now cloned, sequenced, and highly purified. The purification procedures use autoclaved buffers (with 2-mercaptoethanol, DTT, and MgSO4 added, if indicated, after sterilization), and sterile columns, plastic, and glassware. Otherwise, the purification steps are carried out at 4°. During sonication, the extract is kept in a salt-ice water bath, and the sonication interrupted periodically to maintain the temperature below 8°. Extracts and intermediate fractions are frozen in dry ice and stored at -80° if there is a delay in going to the next step. The final purified proteins are stored in small aliquots at -80°. Methods to assay and purify the T4 replication proteins from T4 infected E. coli and from E. coli with expression plasmids are developed in several laboratories.