Human p53 Binds Holliday Junctions Strongly and Facilitates Their Cleavage

Abstract

Holliday junctions in DNA are generated as a product of homologous recombination events. To test the hypothesis that human p53 may bind to Holliday junctions, synthetic junctions with four approximately 75-base pair (Hol75) or approximately 565-base pair (Hol565) arms were generated. As seen by electron microscopy, under conditions in which 50-61% of the Hol565 DNAs were bound by p53, 80-96% of the p53 was located specifically at the junction with, in the latter case, only 4% of the p53 visualized at the DNA ends or along the arms. Given the large number of potential binding sites, this represents very high specificity for the junctions. Gel retardation assays using the Hol75 DNA confirm these observations, and indicate that the tight junction complexes have a half-life of greater than 4 h. The binding of p53 to three-way junctions is severalfold less than to four-way junctions. Addition of p53 greatly increases the rate of resolution of the Hol75 DNA by T4 endonuclease VII and T7 endonuclease I, two enzymes known to cleave such junctions. This latter finding further confirms the interaction of p53 with Holliday junctions and suggests that p53 binding facilitates their resolution in vivo.