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Frame-shift mediated reduction of gain-of-function p53 R273H and deletion of the R273H C-terminus in breast cancer cells result in replication-stress sensitivity.

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.

SUBMITTER: Ellison V

PROVIDER: S-EPMC8202772 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundReporter methods to quantitatively measure the efficiency and specificity of genome editing tools are important for the development of novel editing techniques and successful applications of available ones. However, the existing methods have major limitations in sensitivity, accuracy, and/or readiness for in vivo applications. Here, we aim to develop a straight-forward method by using nucleotide insertion/deletion resulted from genome editing. In this system, a target sequence with frame-shifting length is inserted after the start codon of a cerulean fluorescence protein (CFP) to inactivate its fluorescence. As such, only a new insertion/deletion event in the target sequence will reactivate the fluorescence. This reporter is therefore termed as "Insertion/deletion-activated frame-shift fluorescence protein". To increase its traceability, an internal ribosome entry site and a red fluorescence protein mCherryFP are placed downstream of the reporter. The percentage of CFP-positive cells can be quantified by fluorescence measuring devices such as flow cytometer as the readout for genome editing frequency.ResultsTo test the background noise level, sensitivity, and quantitative capacity of this new reporter, we applied this approach to examine the efficiency of genome editing of CRISPR/Cas9 on two different targeting sequences and in three different cell lines, in the presence or absence of guide-RNAs with or without efficiency-compromising mutations. We found that the insertion/deletion-activated frame-shift fluorescence protein has very low background signal, can detect low-efficiency genome editing events driven by mutated guideRNAs, and can quantitatively distinguish genome editing by normal or mutated guideRNA. To further test whether the positive editing event detected by this reporter indeed correspond to genuine insertion/deletion on the genome, we enriched the CFP-positive cells to examine their fluorescence under confocal microscope and to analyze the DNA sequence of the reporter in the genome by Sanger sequencing. We found that the positive events captured by this reporter indeed correlates with genuine DNA insertion/deletion in the expected genome location.ConclusionThe insertion/deletion-activated frame-shift fluorescence protein reporter has very low background, high sensitivity, and is quantitative in nature. It will be able to facilitate the development of new genome editing tools as well as the application of existing tools.

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Frame-shift mediated reduction of gain-of-function p53 R273H and deletion of the R273H C-terminus in breast cancer cells result in replication-stress sensitivity.

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