Cancer is life-threatening disease with no definitive treatment. Most chemotherapeutic agents take the advantage of the fact that cancer cells have uncontrolled replication and proliferation. In this study we investigated the effect of a Holliday junction (HJ) binding peptide on the prostate cancer cell lines. Prostate cancer (PCa) is one of the major causes of cancer-related death in the United States. The onset of the disease is related to the activation of the testosterone hormone (androgen) receptor upon binding of its ligand. Initial treatment for PCa is androgen deprivation, which works for 12-15 months. In most cases the disease relapses with the tumors becoming androgen-independent, a stage called castrate refraction. Effective treatments are in need for treating the castrate refractory PCa. Our lab has identified peptides which stabilized the HJs formed during phage lambda recombination, by inhibit their resolution. HJs are intermediate steps of the homologous recombination repair (HRR) in both prokaryotes and eukaryotes. As cancer cells depend more on HRR than normal cells, we hypothesize that peptide would decrease cell survival by binding to the HJ intermediates of HRR and blocking repair. We found that peptide, wrwycr causes dose dependent PCa cell death singly or additively in combination with doxorubicin. Exploring the mechanism behind cell death we found that peptide accumulates double stranded DNA breaks in a dose and time dependent manner, as evident from the activation of _H2AX, an early signaling molecule activated with double-strand (ds) DNA breaks. Ds DNA breaks persist as long as 48 h. This leads to the activation of the cell cycle check point proteins (Chk1 and Chk2), arresting the cellular progression to the G2/M-phase of the cell cycle.