Abstract
We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1. The missense R273H GOF mtp53 has a mutated central DNA binding domain that renders it unable to bind specifically to DNA, but maintains the capacity to interact tightly with chromatin. Both the C-terminal domain (CTD) and oligomerization domain (OD) of GOF mtp53 proteins are intact and it is unclear whether these regions of mtp53 are responsible for chromatin-based DNA replication activities. We generated MDA-MB-468 cells with CRISPR-Cas9 edited versions of the CTD and OD regions of mtp53 R273H. These included a frame-shift mtp53 R273Hfs387, which depleted mtp53 protein expression; mtp53 R273HΔ381-388, which had a small deletion within the CTD; and mtp53 R273HΔ347-393, which had both the OD and CTD regions truncated. The mtp53 R273HΔ347-393 existed exclusively as monomers and disrupted the chromatin interaction of mtp53 R273H. The CRISPR variants proliferated more slowly than the parental cells and mt53 R273Hfs387 showed the most extreme phenotype. We uncovered that after thymidine-induced G1/S synchronization, but not hydroxyurea or aphidicholin, R273Hfs387 cells displayed impairment of S-phase progression while both R273HΔ347-393 and R273HΔ381-388 displayed only moderate impairment. Moreover, reduced chromatin interaction of MCM2 and PCNA in mtp53 depleted R273Hfs387 cells post thymidine-synchronization revealed delayed kinetics of replisome assembly underscoring the slow S-phase progression. Taken together our findings show that the CTD and OD domains of mtp53 R273H play critical roles in mutant p53 GOF that pertain to processes associated with DNA replication.
Highlights
The p53 tumor suppressor protein is well known as a transcription factor but p53 has transcription independent functions [1]
Cas9-single guide RNA (sgRNA) targeted alteration of the C-terminal oligomerization (OD) and C-terminal regulatory domain (CTD) of mtp53 R273H within triple negative breast cancer (TNBC) MDA-MB-468 cells results in novel frame-shift and deletion variant expressing cell lines
The amino and carboxyl regions of GOF mtp53 are unchanged for most GOF mtp53 proteins [11]
Summary
The p53 tumor suppressor protein is well known as a transcription factor but p53 has transcription independent functions [1]. One of these functions involves regulation of DNA replication. While purified wtp is able to block SV40 replication in vitro, purified tumor-derived missense mutant p53 (mtp53) proteins do not block SV40 origin of replication (ori) DNA replication or SV40 T-antigen mediated DNA unwinding [10]. While tumorderived missense mtp proteins have altered functions they contain the two N-terminal transactivation domains, followed by a proline rich domain, an altered central DNA binding domain, and the oligomerization domain (OD) and the C-terminal regulatory domain (CTD) [11]
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