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Isolation of recombinant DNA elongation proteins


van Loon, B; Ferrari, E; Hübscher, U (2009). Isolation of recombinant DNA elongation proteins. Methods in Molecular Biology, 521:345-359.

Abstract

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.

Abstract

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Institute of Veterinary Biochemistry and Molecular Biology
Dewey Decimal Classification:570 Life sciences; biology
Date:2009
Deposited On:02 Mar 2010 14:22
Last Modified:05 Apr 2016 14:01
Publisher:Springer
ISSN:1064-3745
PubMed ID:19563116
Other Identification Number:ISBN 978-1-603-27814-0

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