Nucleic Acids Research Advance Access originally published online on February 18, 2007
Nucleic Acids Research 2007 35(5):1624-1637; doi:10.1093/nar/gkm006
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Nucleic Acids Research, 2007, Vol. 35, No. 5 1624-1637
© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
The C-terminal domain of yeast PCNA is required for physical and functional interactions with Cdc9 DNA ligase
1Radiation Oncology Research Laboratory, Department of Radiation Oncology and The Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201-1509, USA, 2Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA and 3Ludwig Institute for Cancer Research, Cancer Center, and Department of Medicine, University of California San Diego School of Medicine, La Jolla, CA 92093-0660, USA
*To whom correspondence should be addressed. Tel: +1 858 784 8119; Fax: +1 585 784 2289; Email: jat{at}scripps.edu Correspondence may also be addressed to Alan Tomkinson. Tel: +1 410 706 2365; Fax: +1 410 706 3000; E-mail: atomkinson{at}som.umaryland.edu
Received November 30, 2006. Revised December 21, 2006. Accepted December 22, 2006.
There is compelling evidence that proliferating cell nuclear antigen (PCNA), a DNA sliding clamp, co-ordinates the processing and joining of Okazaki fragments during eukaryotic DNA replication. However, a detailed mechanistic understanding of functional PCNA:ligase I interactions has been incomplete. Here we present the co-crystal structure of yeast PCNA with a peptide encompassing the conserved PCNA interaction motif of Cdc9, yeast DNA ligase I. The Cdc9 peptide contacts both the inter-domain connector loop (IDCL) and residues near the C-terminus of PCNA. Complementary mutational and biochemical results demonstrate that these two interaction interfaces are required for complex formation both in the absence of DNA and when PCNA is topologically linked to DNA. Similar to the functionally homologous human proteins, yeast RFC interacts with and inhibits Cdc9 DNA ligase whereas the addition of PCNA alleviates inhibition by RFC. Here we show that the ability of PCNA to overcome RFC-mediated inhibition of Cdc9 is dependent upon both the IDCL and the C-terminal interaction interfaces of PCNA. Together these results demonstrate the functional significance of the ß-zipper structure formed between the C-terminal domain of PCNA and Cdc9 and reveal differences in the interactions of FEN-1 and Cdc9 with the two PCNA interfaces that may contribute to the co-ordinated, sequential action of these enzymes.
Present address: Kristina H. Schmidt, Department of Biology, University of South Florida, 4202 E. Fowler Avenue, SCA110, Tampa, FL 33620 USA The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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