Isolation of recombinant DNA elongation proteins

Abstract

This chapter summarizes isolation procedures of four recombinant human proteins crucial for DNA replication: (a) the replicative DNA polymerase (pol) δ, (b) proliferating cell nuclear antigen (PCNA), (c) replication protein A (RP-A), and (d) replication factor C (RF-C). Pol δ is a four-subunit enzyme essential for replication of the lagging strand and possibly of the leading strand. PCNA is a central player important for coordination of the complex network of proteins interacting at the replication fork. RP-A is single-strand DNA-binding protein involved in DNA replication, DNA repair, DNA recombination, and checkpoint control. RF-C as a clamp loader is required for loading of PCNA onto double-stranded DNA and therefore enables PCNA-dependent elongation by pol δ and pol ε. To reconstitute the intact pol δ and RF-C, a baculovirus expression system is used, where insect cells are infected with baculoviruses, each coding for one of the four or five subunits of pol δ or RF-C, respectively. We also present two easy methods to isolate the homotrimeric human PCNA and the heterotrimeric human RP-A from an Escherichia coli expression system. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.