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A previous study has indicated that the ΔFHA mutant of CHFR acts as a dominant-negative and can disrupt the checkpoint function of the wild type protein [1].
Collectively, our data show that the checkpoint function of the FHA domain of CHFR is a core component of anti-proliferative properties against the gastrointestinal carcinogenesis.
Our data further suggest that an off-target effect may similarly explain the purported checkpoint function of the Tao1 kinase.
A mitotic checkpoint function of the Aurora B kinase complex, independently of generating unattached kinetochores, has previously been documented in vertebrates and fission yeast [ 24 27].
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By the use of a cell cycle checkpoint assay, we also confirmed that the FHA domain of CHFR plays an important role in initiating a cell cycle arrest at G2/M, indicating a functional link exists between the anti-proliferative effects and checkpoint function of this tumor suppressor protein via this domain.
Rampant oncogene-induced hyperproliferation is transient because it is intrinsically associated with a still ill-characterized alteration of the DNA replication process, which causes the consequent activation of the checkpoint functions of the DNA damage response (DDR).
We find that both the anti-proliferative effects and checkpoint function of CHFR require the FHA domain, whereas the E3 ligase activity of this tumor suppressor relies on the RF and Cys domains.
The functional recovery of the checkpoint function of CHFR was evaluated also in HCT116 cells after the exogenous introduction of wild type and mutant CHFR proteins.
The rad9 ST462,474AA mutation did not lead to an increase of the phenotype of the ddc1 T602A mutant (Supplementary Figure S6A), which completely abolishes the checkpoint function of Dpb11 providing additional evidence that the rad9 ST462,474AA phenotype is DPB11 dependent.
Since the promoter methylation of the CHFR gene was detected in non-invasive adenoma lesions of colon epithelia [44], the associated loss of the checkpoint function of CHFR in these cells may have a significant impact upon the early stages of colon carcinogenesis.
This phenomenon remains currently unexplained but is in line with previously proposed cell cycle checkpoint functions of this protease, that when absent may sensitize cells to apoptosis.
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