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Identifying amino acid residues in the yeast high mobility group protein HMO2 that are important for DNA binding
Author(s) -
Rahman Wedad,
Ray Sreerupa,
Grove Anne
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.701.3
Subject(s) - hmg box , dna clamp , dna , biochemistry , biology , microbiology and biotechnology , dna polymerase , replication protein a , protein–dna interaction , mutagenesis , dna polymerase ii , high mobility group , dna binding site , dna binding protein , mutant , gene , reverse transcriptase , rna , gene expression , promoter , transcription factor
High mobility group (HMGB) proteins are non‐histone nuclear proteins that play a role in DNA repair. The functional roles of one of the HMG domains of the yeast homolog HMO2 were examined by identifying amino acid residues that are involved in DNA intercalation. Through site directed mutagenesis, a valine in the box A domain, predicted to intercalate between DNA base pairs, was substituted with alanine. A binding assay with linear DNA showed that the mutated protein has lower binding affinity compared to wild type HMO2, while both mutant and wild type proteins have similar affinity for supercoiled DNA. The ability of the mutant HMO2 to protect free DNA ends from exonuclease digestion was determined; both wild‐type protein and the mutated protein were able to protect the DNA from exonuclease activity, suggesting that the substitution does not affect the preferred binding to DNA ends characteristic of HMO2. DNA binding assays further showed that both wild‐type and mutant protein bound with only modest preference to DNA with one loop (tandem mismatches), but both bound with significant preference to DNA with two loops and to DNA with one loop and one abasic site. Evidently, the valine in HMO2 box A is important for the protein's ability to bind to linear DNA, while its substitution does not alter the preferred binding of HMO2 to DNA ends or to DNA with distortions.