High affinity interaction of dipteran high mobility group (HMG) proteins 1 with DNA is modulated by COOH-terminal regions flanking the HMG box domain.

J.R Wiśniewski,E Schulze

doi:10.1016/s0021-9258(17)34117-0

J.R Wiśniewski, E Schulze

Open Access

https://doi.org/10.1016/s0021-9258(17)34117-0

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Abstract

The cells of the dipteran insects Chironomus and Drosophila contain high mobility group (HMG) 1 proteins that are homologous to the HMG1 protein of mammals but comprise one instead of two DNA-binding HMG boxes. Mobility shift assays have revealed that Chironomus cHMG1a and cHMG1b bind double strand and four-way junction DNA in a similar way at apparent dissociation constants in the range of 7.5-20 x 10(-9) M. Both proteins are monomeric and highly asymmetric molecules in solution. cHMG1a and cHMG1b exhibit Stokes' radii of 2.4 and 2.3 nm, respectively, and both show a frictional ratio of 1.5. Despite these similarities in their hydrodynamic properties, the binding site of cHMG1a on DNA is approximately 1.5 of the size found for the cHMG1b. Enzymatically and chemically prepared peptides of cHMG1a as well as bacterially expressed cHMG1a with terminal deletions and point substitutions showed that sequences flanking the folded domain that constitutes the HMG box are essential for the interaction of the HMG box with DNA. In particular, changes in the number of positive and negative charges, respectively, within basic and acidic domains modulated the DNA binding affinity of the cHMG1a protein. The alteration of fluorescence of the Trp residues suggest that this modulation is due to interaction of the acidic domain with the positively charged HMG box.

Highlights

Mu8 cHMGla and cHMGlbbind double strand and four-molecules have only one folded domain that is followed by a way junction DNA in a similawray at apparent dissociationconstantsintherange of 7.5-20 x lo-’ M
The HMGlA proteins interact quences flanking the folded domain that constitutes ptrheeferentially with negatively supercoiled DNA (Sheflin and HMG box are essential for the interaction of the HMG Spaulding, 1989; Sheflinet al.,1993),AT-rich DNA(Brown and box with DNA
The bases before cHMGla, Chironomus HMG protein la; cHMGlb, Chironomus HMG the start codons, before the internal Met-12 codon, and before an artiprotein lb; HPLC, high performance liquid chromatography;PAGE, ficially created Met codon at position 7 were changed to the sequence polyacrylamide gel electrophoresis;PCR,polymerasechainreaction; CAT, so that the resulting sequence CATATG could be recognized by bp, base pairs

Summary

EXPERIMENTAL PROCEDURES

The abbreviationsused are: HMG1, high mobility group protein 1; ends of the coding regions of the cDNA clones pWS5.1 (cHMGla) and HMG2,highmobilitygroupprotein. The bases before cHMGla, Chironomus HMG protein la; cHMGlb, Chironomus HMG the start codons, before the internal Met-12 codon, and before an artiprotein lb; HPLC, high performance liquid chromatography;PAGE, ficially created Met codon at position 7 were changed to the sequence polyacrylamide gel electrophoresis;PCR,polymerasechainreaction; CAT, so that the resulting sequence CATATG could be recognized by bp, base pairs. Oligonucleotides used forthe construction of 3'-ends of the coding purified by HPLC, and subsequentloyne of the strands was labeled with regions are: cHMGla and cHMGlb, GTAAAACGACGGCCAGT TCTTCATCTGATTCGTC'ITCCTCCTCAT.PCR was performed with 40 ried out on aPerkin-Elmer LS-50 spectrofluorometer.Theemission p~ dNTP, 1p~ of each primer, 1unit of Taq DNA polymerase (Amer- spectrawererecordedusing2.5and4nmslits, for excitationand sham, United Kingdom)/5O pl, and 1 x Taq buffer from the enzyme emission, respectively.

RESULTS

Protein Fluorescence and Its Quenching upon DNA

DISCUSSION