DETERMINANTS OF DNA-BINDING AND BENDING BY THE SACCHAROMYCES-CEREVISIAE HIGH-MOBILITY GROUP PROTEIN NHP6A THAT ARE IMPORTANT FOR ITS BIOLOGICAL-ACTIVITIES - ROLE OF THE UNIQUE N-TERMINUS AND PUTATIVE INTERCALATING METHIONINE

The non-histone proteins 6A/B (NHP6A/B) of Saccharomyces cerevisiae ar e high mobility group proteins that bind and severely bend DNA of mixe d sequence. They exhibit high affinity for linear DNA and even higher affinity for microcircular DNA. The 16-amino acid basic segment locate d N-terminal to the high mobility group domain is required for stable complex formation on both linear and microcircular DNA. Although mutan ts lacking the N terminus are able to promote microcircle formation an d Hin invertasome assembly at high protein concentrations, they are un able to form stable complexes with DNA, co-activate transcription, and complement the growth defect of Delta nhp6a/b mutants. A basic patch between amino acids 13 and 16 is critical for these activities, and a second basic patch between residues 8 and 10 is required for the forma tion of monomeric complexes with linear DNA. Mutational analysis sugge sts that proline 18 may direct the path of the N-terminal arm to facil itate DNA binding, whereas the conserved proline at position 21, tyros ine 28, and phenylalanine 31 function to maintain the tertiary structu re of the high mobility group domain. Methionine 29, which may interca late into DNA, is essential for NHP6A-induced micro-circle formation o f 75-bp but not 98-bp fragments in vitro, and for full growth compleme ntation of Delta nhp6a/b mutants in vivo.