Abstract The tumor suppressor protein p53 is critical for cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds DNA response elements to regulate transcription of target genes. p53 response elements consist of two decameric half-sites, and data suggest one p53 dimer in the tetramer binds to each half-site. Despite a broad literature describing p53 binding DNA, unanswered questions remain, due partly to the need for more quantitative and structural studies with full length protein. Here we describe a single molecule fluorescence system to visualize full length p53 tetramers binding DNA in real time. The data revealed a dynamic interaction in which tetrameric p53/DNA complexes assembled and disassembled without a dimer/DNA intermediate. On a wild type DNA containing two half sites, p53/DNA complexes existed in two kinetically distinct populations. p53 tetramers bound response elements containing only one half site to form a single population of complexes with reduced kinetic stability. Altering the spacing and helical phasing between two half sites affected both the population distribution of p53/DNA complexes and their kinetic stability. Our real time single molecule measurements of full length p53 tetramers binding DNA reveal the parameters that define the stability of p53/DNA complexes, and provide insight into the pathways by which those complexes assemble.
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Nanoscale Interaction of Endonuclease APE1 with DNA
Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA repair and transcriptional regulation mechanisms. This multifunctional activity of APE1 should be supported by specific structural properties of APE1 that have not yet been elucidated. Herein, we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA containing two well-separated G-rich segments. Complexes of APE1 with DNA containing G-rich segments were visualized, and analysis of the complexes revealed the affinity of APE1 to G-rich DNA sequences, and their yield was as high as 53%. Furthermore, APE1 is capable of binding two DNA segments leading to the formation of loops in the DNA–APE1 complexes. The analysis of looped APE1-DNA complexes revealed that APE1 can bridge G-rich segments of DNA. The yield of loops bridging two G-rich DNA segments was 41%. Analysis of protein size in various complexes was performed, and these data showed that loops are formed by APE1 monomer, suggesting that APE1 has two DNA binding sites. The data led us to a model for the interaction of APE1 with DNA and the search for the specific sites. The implication of these new APE1 properties in organizing DNA, by bringing two distant sites together, for facilitating the scanning for damage and coordinating repair and transcription is discussed.
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- Award ID(s):
- 1941049
- PAR ID:
- 10563751
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- International Journal of Molecular Sciences
- Volume:
- 25
- Issue:
- 10
- ISSN:
- 1422-0067
- Page Range / eLocation ID:
- 5145
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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