Distinct Regions of Cu(I)·ACE1 Contact Two Spatially Resolved DNA Major Groove Sites

Albert Dobi,Charles T Dameron,Stella Hu,Dean Hamer,Dennis R Winge

doi:10.1074/jbc.270.17.10171

Albert Dobi, Charles T Dameron + Show 3 more

Open Access

https://doi.org/10.1074/jbc.270.17.10171

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Abstract

The interaction between the Cu(I).ACE1 (CuACE1) transcription factor and its DNA binding site in the yeast metallothionein gene was studied by systematically altering the DNA sequence through base substitution, modification, and deletions as well as by altering the protein structure through chemical modification. We show here that CuACE1 is comprised of two distinct domains that contact DNA through minor groove interactions located between two major groove interaction sites. The minor groove interactions are shown to be critical for formation of a stable CuACE1.DNA complex. The NH2-terminal segment of ACE1 is shown to contact the 5'-most distal major groove site.

Highlights

The interaction between the Cu(l)·ACEI (CuACEl) transcription factor and its DNA binding site in the yeast metallothionein gene was studied by systematically altering the DNA sequence through base substitution, modification, and deletions as well as by altering the protein structure through chemical modification
In addition to the known major groove contacts at each end of the half-site, we demonstrate that CuACEl makes specific minor groove contacts that are important for stabilization of the CuACE 1· DNA complex
Th e minor groov e a nd proxima l major groove si tes are essentia l for high a ffinity binding of CuACE l, whereas th e distal site contributes less to t he ene rgy of stabiliz ation of t he complex

Summary

Current address

National Research Centre for Environmental Toxicology, University of Queensland, Brisbane 4072, Australia. CuACEl is known to preferentially bind the left half-site as opposed to the right half-site of the UASc palindrome in the CUPI metallothionein gene [11]. Previous footprinting studies suggested that CuACEl crosses a minor groove to make major groove contacts at each end of the half-site [23]. In the present work we assessed the base-specific interactions between CuACEl and a chemically modified CuACEl with the left half-site of the DAS c sequence. In addition to the known major groove contacts at each end of the half-site, we demonstrate that CuACEl makes specific minor groove contacts that are important for stabilization of the CuACE 1· DNA complex. The chemically modified CuACEl was found to be incapable of binding to the distal major groove site of the DAS c half-site, whereas the minor and proximal major groove interactions remained unchanged

EXPERIMENTAL PROCEDURES

I 1I 11111I 1I 1I I 1111111111

B UASc L ft Arm

Findings

DISCUSSION