Characterization of the DNA binding and structural properties of the BRCT region of human replication factor C p140 subunit.
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 approximately 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 alpha-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 alpha-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 (1)H NMR and CD spectroscopy. Mutation of weakly conserved residues on one face of the N-terminal alpha-helix and of residues within the cluster disrupted DNA binding, suggesting a likely binding interface on the protein.
Kobayashi
Masakazu
M
Leiden Institute of Chemistry, Leiden University, The Netherlands.
Figaroa
Francis
F
Meeuwenoord
Nico
N
Jansen
Lars E T
LE
Siegal
Gregg
G
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D051818:Replication Protein C / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D021122:Protein Subunits; D004247:DNA; D051818:Replication Protein C