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The N‐terminal Primase‐like Domain of the Drosophila Mitochondrial Replicative DNA Helicase Binds an Iron Sulfur Cluster: Structural and Functional Implications
Author(s) -
Farnum Gregory A,
Kaguni Laurie S
Publication year - 2012
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.26.1_supplement.739.6
Subject(s) - primase , helicase , biology , biochemistry , dna , binding site , microbiology and biotechnology , biophysics , gene , rna , reverse transcriptase
The N‐terminal region of the metazoan mitochondrial replicative DNA helicase (mtDNA helicase) shares extensive sequence conservation with the primase domain of bacteriophage T7 primase‐helicase. Primase activity has not been demonstrated in metazoan mtDNA helicases, most likely because several Mg 2+ ‐binding residues of the primase active site in T7 primase are not conserved in Metazoa, although several conserved primase sequence motifs are well conserved. Interestingly, insects have retained the four cysteines of conserved motif I that have been shown to bind zinc in the T7 enzyme. Therefore, to study the function of the N‐terminal domain of metazoan mtDNA helicase, we designed two N‐terminal constructs of the Drosophila melanogaster protein comprising residues N24‐A333 (NTD) and N24‐P123 (ZBD). Purified fractions of the full N‐terminal domain construct (NTD) contain an iron sulfur cluster, as determined by UV‐Visible spectroscopy, as well as by iron and sulfide determination. Based on T7 homology, the NTD contains the conserved zinc binding (ZBD) and RNA polymerase (RPD) domains. Purified fractions of the ZBD construct also contained an Fe‐S cluster, suggesting the zinc‐binding site has been converted to an Fe‐S binding site in the Dm mtDNA helicase. We will present data using differential scanning fluorimetry, fluorescence resonance energy transfer and differential scanning calorimetry regarding the involvement of the Fe‐S cluster in the structural stability and in nucleic acid ligand binding by the NTD form.