The C-terminus of gain-of-function mutant p53 R273H is required for association with PARP1 and Poly-ADP-Ribose release_beipob5vc5gztbfnocg7jr2com

Abstract

<jats:title>Abstract</jats:title> The TP53 gene is mutated in 80% of triple-negative breast cancers. Cells that harbor the hot-spot p53 gene mutation R273H produce an oncogenic mutant p53 (mtp53) that enhances cell proliferative and metastatic properties. The enhanced activities of mtp53 are collectively referred to as gain-of-function (GOF), and may include transcription-independent chromatin-based activities shared with wild type p53 (wtp53) such as association with replicating DNA and DNA replication associated proteins like Poly-ADP-Ribose Polymerase 1 (PARP1). However how mtp53 up-regulates cell proliferation, is not well understood. Wild-type p53 interacts with PARP1 using a portion of its C-terminus. The wtp53 oligomerization (OD) and far C-terminal domain (CTD) located within the C-terminus constitute putative GOF-associated domains, since R273H breast cancer cells lacking both domains manifest slow proliferation phenotypes. We addressed if the C-terminal region of mtp53 R273H is important for chromatin interaction and breast cancer cell proliferation using CRISPR-Cas9 mutated MDA-MB-468 cells endogenously expressing mtp53 R273H C-terminal deleted isoforms (R273HΔ381-388 and R273HΔ347-393). The mtp53 R273HΔ347-393 lacks the CTD and a portion of the OD. We observed that cells harboring mtp53 R273HΔ347-393 (compared to mtp53 R273H full-length) manifest a significant reduction in chromatin, PARP1, Poly-ADP-Ribose (PAR), and replicating DNA binding. These cells also exhibited impaired response to hydroxyurea (HU) replicative stress, decreased sensitivity to the PARP-trapping drug combination temozolomide-talazoparib, and increased phosphorylated 53BP1 foci, suggesting reduced Okazaki fragment processing. Implications: The C-terminal region of mtp53 confers GOF activity that mediates mtp53-PARP1 and PAR interactions assisting DNA replication, thus implicating new biomarkers for PARP inhibitor therapy.
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