Probing the DNA sequence specificity of Escherichia coli RECA protein.
Escherichia coli RecA protein catalyzes the central DNA strand-exchange step of homologous recombination, which is essential for the repair of double-stranded DNA breaks. In this reaction, RecA first polymerizes on single-stranded DNA (ssDNA) to form a right-handed helical filament with one monomer per 3 nt of ssDNA. RecA generally binds to any sequence of ssDNA but has a preference for GT-rich sequences, as found in the recombination hot spot Chi (5'-GCTGGTGG-3'). When this sequence is located within an oligonucleotide, binding of RecA is phased relative to it, with a periodicity of three nucleotides. This implies that there are three separate nucleotide-binding sites within a RecA monomer that may exhibit preferences for the four different nucleotides. Here we have used a RecA coprotease assay to further probe the ssDNA sequence specificity of E.coli RecA protein. The extent of self-cleavage of a lambda repressor fragment in the presence of RecA, ADP-AlF4 and 64 different trinucleotide-repeating 15mer oligonucleotides was determined. The coprotease activity of RecA is strongly dependent on the ssDNA sequence, with TGG-repeating sequences giving by far the highest coprotease activity, and GC and AT-rich sequences the lowest. For selected trinucleotide-repeating sequences, the DNA-dependent ATPase and DNA-binding activities of RecA were also determined. The DNA-binding and coprotease activities of RecA have the same sequence dependence, which is essentially opposite to that of the ATPase activity of RecA. The implications with regard to the biological mechanism of RecA are discussed.
Duke Scholars
Altmetric Attention Stats
Dimensions Citation Stats
Published In
DOI
EISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Viral Regulatory and Accessory Proteins
- Viral Proteins
- Repressor Proteins
- Repetitive Sequences, Nucleic Acid
- Rec A Recombinases
- Protein Binding
- Oligonucleotides
- Escherichia coli Proteins
- Escherichia coli
- Developmental Biology
Citation
Published In
DOI
EISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Viral Regulatory and Accessory Proteins
- Viral Proteins
- Repressor Proteins
- Repetitive Sequences, Nucleic Acid
- Rec A Recombinases
- Protein Binding
- Oligonucleotides
- Escherichia coli Proteins
- Escherichia coli
- Developmental Biology