REGION OF INSECT HIGH-MOBILITY GROUP (HMG)-1 PROTEIN HOMOLOGOUS TO HELIX-2 OF THE RAT HMG1-B BOX IS IN CLOSE CONTACT WITH DNA

The dipteran insects Chironomus and Drosophila have high mobility grou p (HMG) 1 proteins that are similar to mammalian HMG1 but contain one instead of two HMG1 boxes. The interaction of the Chironomus HMG1 prot eins cHMG1a and cHMG1b with double-stranded and four-way junction DNA was analyzed by investigating the accessibility of defined sequences o f the HMG1 box to specific antibodies within the DNA-protein complex i n vitro. Antibody epitopes located in the three helices and in the tur n between helices 1 and 2 of the HMG1 box were identified on a membran e onto which 90 decapeptides with overlapping sequences spanning the e ntire sequence of cHMG1a had been bound. Monospecific antibodies direc ted against selected epitopes were purified from a polyclonal antiseru m by affinity chromatography. Helices 1 and 3 as well as the turn betw een helices 1 and 2 were found 60 be accessible to antibodies in the c omplex. In contrast, antibodies recognizing an epitope on putative hel ix 2 of cHMG1a and cHMG1b were unable to produce supershifts on gels o f the DNA-protein complexes with both DNAs. These data suggest that he lix 2 of the HMG1 box of proteins cHMG1a and cHMG1b is essentially res ponsible for contacts with DNA.