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The tumor suppressor adenomatous polyposis coli (APC) is implicated in regulating multiple stages of the cell cycle. APC participation in G1/S is attributed to its recognized role in Wnt signaling. APC function in the G2/M transition is less well established. To identify novel protein partners of APC that regulate the G2/M transition, APC was immunoprecipitated from colon cell lysates and associated proteins were analyzed by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF). Topoisomerase IIalpha (topo IIalpha) was identified as a potential binding partner of APC. Topo IIalpha is a critical regulator of G2/M transition. Evidence supporting an interaction between endogenous APC and topo IIalpha was obtained by coimmunoprecipitation, colocalization, and Förster resonance energy transfer (FRET). The 15-amino acid repeat region of APC (M2-APC) interacted with topo IIalpha when expressed as a green fluorescent protein (GFP)-fusion protein in vivo. Although lacking defined nuclear localization signals (NLS) M2-APC predominantly localized to the nucleus. Furthermore, cells expressing M2-APC displayed condensed or fragmented nuclei, and they were arrested in the G2 phase of the cell cycle. Although M2-APC contains a beta-catenin binding domain, biochemical studies failed to implicate beta-catenin in the observed phenotype. Finally, purified recombinant M2-APC enhanced topo IIalpha activity in vitro. Together, these data support a novel role for APC in the G2/M transition, potentially through association with topo IIalpha.