P53 Monitors Replication Fork Regression by Binding to “Chickenfoot” Intermediates

Deepa Subramanian,Jack D. Griffith

doi:10.1074/jbc.m506348200

Deepa Subramanian, Jack D. Griffith

Open Access

https://doi.org/10.1074/jbc.m506348200

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Abstract

The tumor suppressor protein, p53, utilizes multiple mechanisms to ensure faithful transmission of the genome including regulation of DNA replication, repair, and recombination. Monitoring these pathways may involve direct binding of p53 to the DNA intermediates of these processes. In this study, we generated templates resembling stalled replication forks and utilized electron microscopy to examine p53 interactions with these substrates. Our results show that p53 bound with high affinity to the junction of stalled forks, whereas two cancer-derived p53 mutants showed weak binding. Additionally, some of the templates were rearranged to form "chickenfoot" structures in the presence of p53. These were mostly formed due to p53 trapping intermediates of spontaneous fork regression; however, in a small population, the protein appeared to be promoting their formation. Collectively, these results demonstrate the importance of sequence-independent binding in p53-mediated maintenance of genomic integrity.

Highlights

Several reports have shown that wild type p53 acts as a negative regulator of spontaneous as well as radiation-induced homologous recombination [13,14,15]
In this study we have shown that p53 binds with high affinity to the junctions of stalled replication forks
By electron microscopy (EM), we were able to observe that a fraction of the p53-bound replication forks were rearranged to form four-stranded chickenfoot structures

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—The 400-bp G-less cassette [29] was cloned into the EcoRI and BamHI sites of pBluescript KS (plasmid pBSGLess). Single strand tails were created by strand displacement using 5 units of the Klenow fragment (exoϪ) (New England Biolabs) in 20-␮l reactions containing 100 mM Tris, pH 7.5, 50 mM MgCl2, 0.1 M dithiothreitol, and 5 mM each dATP, dTTP, and dGTP at 37 °C for 30 min. To create a double strand tail, primer 25 (5Ј-CTTCCTCCATCTATACCACC-3Ј) was annealed to the 3Ј-end of the displaced single strand at a 10-fold molar excess at 37 °C for 30 min in 40-␮l reactions containing 100 mM Tris, pH 7.5, 50 mM MgCl2, 0.1 M dithiothreitol, and 5 mM each dATP, dTTP, and dCTP followed by the addition of 5 units of Klenow fragment (exoϪ) and further incubation at 37 °C for 30 min.

RESULTS

TABLE TWO

Tail position

TABLE THREE

DISCUSSION