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J. Biol. Chem., Vol. 281, Issue 7, 4308-4317, February 17, 2006

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Characterization of the DNA Binding and Structural Properties of the BRCT Region of Human Replication Factor C p140 Subunit^*

From the Leiden Institute of Chemistry, Leiden University, Postbus 9502, 2300 RA Leiden, The Netherlands

BRCT domains, present in a large number of proteins that are involved in cell cycle regulation and/or DNA replication or repair, are primarily thought to be involved in protein-protein interactions. The large (p140) subunit of replication factor C contains a sequence of 100 amino acids in the N-terminal region that binds DNA and is distantly related to known BRCT domains. Here we show that residues 375-480, which include 28 amino acids N-terminal to the BRCT domain, are required for 5'-phosphorylated double-stranded DNA binding. NMR chemical shift analysis indicated that the N-terminal extension includes an -helix and confirmed the presence of a conserved BRCT domain. Sequence alignment of the BRCT region in the p140 subunit of replication factor C from various eukaryotes has identified very few absolutely conserved amino acid residues within the core BRCT domain, whereas none were found in sequences immediately N-terminal to the BRCT domain. However, mapping of the limited number of conserved, surface-exposed residues that were found onto a homology model of the BRCT domain, revealed a clustering on one side of the molecular surface. The cluster, as well as a number of amino acids in the N-terminal -helix, were mutagenized to determine the importance for DNA binding. To ensure minimal structural changes because of the introduced mutations, proteins were checked using one-dimensional ¹H NMR and CD spectroscopy. Mutation of weakly conserved residues on one face of the N-terminal -helix and of residues within the cluster disrupted DNA binding, suggesting a likely binding interface on the protein.

Received for publication, October 12, 2005 , and in revised form, December 16, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Ludwig Institute for Cancer Research, CMM-East 3072, 9500 Gilman Dr., La Jolla, CA 92093.

² To whom correspondence should be addressed. Tel.: 31-71-527-4543; Fax: 31-71-527-4593; E-mail: g.siegal{at}chem.leidenuniv.nl.

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