Characterization of the role of PTIP in the DNA damage response.

Abstract

An effective response to DNA damage requires the ability of damage-associated response proteins to locate to sites of damage, access the DNA, and assemble into a working complex. The mammalian BRCT domain-containing protein PTIP (Pax transactivation domain-interacting protein), encoded by the <italic> PaxIP1</italic> gene (Mouse Genome Informatics), is a binding protein that is specific for phosphorylated substrates of Ataxia Telangiectasia-mutated (ATM), which controls the response to ionizing radiation (IR)-induced damage. An IR-dependent complex that includes the DNA damage response (DDR) proteins 53BP1, Rad50, and Smc1, which are downstream effectors of ATM signaling, was copurified with PTIP from nuclear lysates of irradiated cells. The PTIP null mouse exhibited embryonic lethality between E7 and E10, which corresponds to the time of gastrulation. E7.5 and E8.5 embryo sections revealed abundant apoptosis in the region of the embryonic ectoderm, and E3.5 embryo-derived cells demonstrated a failure to thrive in culture, and radiosensitivity. Mouse embryonic fibroblasts (MEFs) derived from PTIP conditional knockout mice were used to assess the functional consequences of PTIP deficiency at the cellular level. Following adeno-Cre-mediated PTIP inactivation, the null fibroblasts exhibited a lower rate of growth than heterozygous MEFs isolated from littermates. The decline in growth was accompanied by an increase in apoptosis and an increased rate of senescence. Gross DNA repair occurs at a similar rate to wild-type controls, and the duration of the DDR was unaltered as measured by the abundance of gammaH2AX foci. Analysis of metaphase spreads following treatment with low dose IR revealed a significant increase in chromosome breaks in PTIP-null MEFs, compared to wild-type controls. Despite published reports of PTIP deficiency resulting in reduced ATM-dependent cell cycle checkpoint signaling, checkpoint analysis indicated that these checkpoints are intact. We conclude that the increased apoptosis and senescence in PTIP-null MEFs could possibly explain the proliferation defect of these cells, but does not explain the lethality of PTIP deficiency in the whole mouse. We conclude that the contribution of PTIP to the DNA damage response is most likely a role in promoting DNA repair and, possibly, mediation of one or more cell cycle checkpoints.