Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent.
AUTOR(ES)
Ebright, R H
RESUMO
Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of the helix-turn-helix motif. [(N-Acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP binds to a 22-base-pair DNA recognition site with Kobs = 1 x 10(8) M-1. In the presence of Cu(II) and reducing agent, [(N-acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP cleaves DNA at four adjacent nucleotides on each DNA strand within the DNA recognition site. The DNA cleavage reaction has been demonstrated using 40-base-pair and 7164-base-pair DNA substrates. The DNA cleavage reaction is not inhibited by dam methylation of the DNA substrate. Such semisynthetic site-specific DNA cleavage agents have potential applications in chromosome mapping, cloning, and sequencing.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=53797Documentos Relacionados
- A 269-amino-acid segment with a pseudo-leucine zipper and a helix-turn-helix motif codes for the sequence-specific DNA-binding domain of herpes simplex virus type 1 origin-binding protein.
- The helix–turn–helix motif of bacterial insertion sequence IS911 transposase is required for DNA binding
- Improved detection of helix-turn-helix DNA-binding motifs in protein sequences.
- Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif.
- Ribosomal protein L7/L12 has a helix-turn-helix motif similar to that found in DNA-binding regulatory proteins.