Assembling bacteriophage T7 leading-strand replisome for structural investigation.

Replicative helicase and polymerase form the leading-strand replisome that unwinds parental DNA and performs continuous leading-strand DNA synthesis. Uncoupling of the helicase-polymerase complex results in replication stress, replication errors, and genome instability. Although numerous replisomes from different biological systems have been reconstituted and characterized, structural investigations of the leading-strand replisome complex are hindered by its large size and dynamics. We have determined the first replisome structure on a fork substrate with bacteriophage T7 replisome as a model system. Here, we summarized our protocols to prepare and characterize the coupled T7 replisome complex. Similar methods can potentially be applied for structural investigations of more complicated replisomes.