Isolation of recombinant DNA elongation proteins.
This chapter summarizes isolation procedures of four recombinant human proteins crucial for DNA replication: (a) the replicative DNA polymerase (pol) delta, (b) proliferating cell nuclear antigen (PCNA), (c) replication protein A (RP-A), and (d) replication factor C (RF-C). Pol delta 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 delta and pol epsilon. To reconstitute the intact pol delta 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 delta 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.
van Loon
Barbara
B
Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Zürich, Switzerland.
Ferrari
Elena
E
Hübscher
Ulrich
U
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D004258:DNA Polymerase III / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051716:Replication Protein A / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D051818:Replication Protein C / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics
D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; D004258:DNA Polymerase III; D051818:Replication Protein C