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Protocol for in vitro phosphorylation of the MUS81-binding region of SLX4 using CDK1-cyclin B.

ABSTRACT: Phosphorylation is a post-translational modification that can alter protein structure and regulate protein-protein interactions. Here, we present a procedure for in vitro phosphorylation of the MUS81-binding region of SLX4 (SLX4^MBR) using cyclin-dependent kinase 1-cyclin B. We describe steps for the dialysis and phosphorylation of target proteins followed by purification using size-exclusion chromatography. Finally, we detail a system to monitor phosphorylation effectiveness and identify phosphorylated residues. We anticipate this protocol to be readily adapted for other protein targets or kinases. For complete details on the use and execution of this protocol, please refer to Payliss et al. (2022).¹.

SUBMITTER: Payliss BJ

PROVIDER: S-EPMC10025271 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Protocol for in vitro phosphorylation of the MUS81-binding region of SLX4 using CDK1-cyclin B.

Payliss Brandon J BJ Wyatt Haley D M HDM

STAR protocols 20230313 2

Phosphorylation is a post-translational modification that can alter protein structure and regulate protein-protein interactions. Here, we present a procedure for in vitro phosphorylation of the MUS81-binding region of SLX4 (SLX4<sup>MBR</sup>) using cyclin-dependent kinase 1-cyclin B. We describe steps for the dialysis and phosphorylation of target proteins followed by purification using size-exclusion chromatography. Finally, we detail a system to monitor phosphorylation effectiveness and ident ...[more]

PMID: 36917604

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Project description:PurposeClinical proton beam therapy has been based on the use of a generic relative biological effectiveness (RBE) of ∼1.1. However, emerging data have suggested that Fanconi anemia (FA) and homologous recombination pathway defects can lead to a variable RBE, at least in vitro. We investigated the role of SLX4 (FANCP), which acts as a docking platform for the assembly of multiple structure-specific endonucleases, in the response to proton irradiation.Methods and materialsIsogenic cell pairs for the study of SLX4, XPF/ERCC1, MUS81, and SLX1 were irradiated at the mid-spread-out Bragg peak of a clinical proton beam (linear energy transfer 2.5 keV/μm) or with 250 kVp x-rays, and the clonogenic survival fractions were determined. To estimate the RBE of the protons relative to cobalt-60 photons (Co60Eq), we assigned a RBE(Co60Eq) of 1.1 to x-rays to correct the physical dose measured. Standard DNA repair foci assays were used to monitor the damage responses, and the cell cycle distributions were assessed by flow cytometry. The poly(ADP-ribose) polymerase inhibitor olaparib was used for comparison.ResultsLoss of SLX4 function resulted in an enhanced proton RBE(Co60Eq) of 1.42 compared with 1.11 for wild-type cells (at a survival fraction of 0.1; P<.05), which correlated with increased persistent DNA double-strand breaks in cells in the S/G2 phase. Genetic analysis identified the SLX4-binding partner MUS81 as a mediator of resistance to proton radiation. Both proton irradiation and olaparib treatment resulted in a similar prolonged accumulation of RAD51 foci in SLX4/MUS81-deficient cells, suggesting a common defect in the repair of DNA replication fork-associated damage.ConclusionsA defect in the FA pathway at the level of SLX4 results in hypersensitivity to proton radiation, which is, at least in part, due to impaired MUS81-mediated processing of replication forks that stall at clustered DNA damage. In vivo and clinical studies are needed to confirm these findings in human cancers.

Dataset Information

Protocol for in vitro phosphorylation of the MUS81-binding region of SLX4 using CDK1-cyclin B.

Publications

Protocol for in vitro phosphorylation of the MUS81-binding region of SLX4 using CDK1-cyclin B.

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