Mutagenesis of the HMGB (high-mobility group B) protein Cmb1 (cytosine-mismatch binding 1) of Schizosaccharomyces pombe: effects on recognition of DNA mismatches and damage.
Cmb1 (cytosine-mismatch binding 1) is a high-mobility group (HMG) protein of Schizosaccharomyces pombe, which consists of 223 amino acids and has a single HMG domain at the C-terminal end. We have created several mutant and deletion forms of the Cmb1 protein and studied the effects on general DNA binding and specific binding to DNA mismatches and damaged DNA. Cmb1Delta41 (i.e. Cmb1 from which the 41 N-terminal amino acids have been deleted) bound specifically to cytosine-containing mismatches, to the cisplatin-induced intrastrand cross-links cis -GG and cis -AG and to an O (6)-methylguanine lesion. DNA binding was not affected when the 45 N-terminal amino acids were deleted, but was abolished in the absence of the 50 N-terminal amino acids, and was reduced when Cmb1 was truncated by between five and eleven C-terminal amino acids. Cmb1, both with and without the C-terminal truncations, retained its DNA binding affinity after heating at 95 degrees C. The cmb1 gene was induced when S. pombe cells were treated with cisplatin. Mitotic mutation rates were increased in a S. pombe cmb1 null mutant and in a cmb1-(1-212) mutant, which encodes a Cmb1 protein lacking the 11 C-terminal amino acids. We conclude that mutation avoidance by Cmb1 is distinct from Msh2-dependent mismatch repair, but related to nucleotide excision repair.
Kunz
Christophe
C
Institute of Cell Biology, University of Bern, Baltzerstrasse 4, Switzerland.
Zurbriggen
Karin
K
Fleck
Oliver
O
D000970:Antineoplastic Agents / Q000494:pharmacology; D020137:Base Pair Mismatch; D015152:Blotting, Northern; D002945:Cisplatin / Q000494:pharmacology; D003596:Cytosine / Q000378:metabolism; D004249:DNA Damage; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006147:Guanine / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D008938:Mitosis / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D012568:Schizosaccharomyces / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins
D000970:Antineoplastic Agents; C115714:Cmb1 protein, S pombe; D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D006609:High Mobility Group Proteins; D029702:Schizosaccharomyces pombe Proteins; D006147:Guanine; D003596:Cytosine; D002945:Cisplatin