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High affinity insertion/deletion lesion binding by p53. Evidence for a role of the p53 central domain
1Department of Biochemistry, Center in Molecular Toxicology and The Vanderbilt Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Abstract:
In addition to binding DNA in a sequence-specific manner, p53 can interact with nucleic acids in a sequence-independent manner. p53 can bind short single-stranded DNA and double-stranded DNA containing nucleotide loops; these diverse associations may be critical for p53 signal transduction. In this study, we analyzed p53 binding to DNA fragments containing insertion/deletion mismatches (IDLs). p53 required an intact central domain and dimerization domain for high affinity complex formation with IDLs. In fact, the C terminus of p53 (amino acids 293-393) was functionally replaceable with a foreign dimerization domain in IDL binding assays. From saturation binding studies we determined that the KD of p53 binding to IDLs was 45 pM as compared with a KD of 31 pM for p53 binding to DNA fragments containing a consensus binding site. Consistent with these dissociation constants, p53-IDL complexes were dissociated with relatively low concentrations of competitor consensus site-containing DNA. Although p53 has a higher affinity for DNA with a consensus site as compared with IDLs, the relative number and availability of each form of DNA in a cell immediately after DNA damage may promote p53 interaction with DNA lesions. Understanding how the sequence-specific and nonspecific DNA binding activities of p53 are integrated will contribute to our knowledge of how signaling cascades are initiated after DNA damage.
Insights
The tumor suppressor protein p53 binds DNA with both sequence-specific and non-specific interactions. This study reveals p53
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The p53 protein plays a crucial role in cellular responses to DNA damage.
- p53 exhibits both sequence-specific and sequence-independent DNA binding capabilities.
- Understanding p53's DNA interactions is key to deciphering DNA damage signaling pathways.
Purpose of the Study:
- To investigate the binding of p53 to DNA fragments containing insertion/deletion mismatches (IDLs).
- To identify the domains of p53 essential for high-affinity binding to IDLs.
- To compare the binding affinity of p53 to IDLs versus canonical DNA binding sites.
Main Methods:
- Saturation binding studies were employed to quantify p53-DNA interactions.
- Analysis of p53 binding to DNA fragments with varying structures, including IDLs.
- Functional replacement assays to assess the role of specific p53 domains in IDL binding.
Main Results:
- p53 requires intact central and dimerization domains for high-affinity binding to IDLs.
- The C-terminus of p53 can be functionally replaced by a foreign dimerization domain for IDL binding.
- p53 demonstrated a high affinity for IDLs (KD = 45 pM), comparable to its binding to consensus sites (KD = 31 pM).
Conclusions:
- p53 binds to DNA lesions like IDLs with significant affinity.
- Both sequence-specific and non-specific DNA binding modes contribute to p53's function after DNA damage.
- The interplay between different DNA binding activities of p53 is crucial for initiating cellular responses to DNA damage.