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USP11 mediates repair of DNA-protein cross-links by deubiquitinating SPRTN metalloprotease.

ABSTRACT: DNA-protein cross-links (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription, and recombination. USP11 deubiquitinase participates in DNA repair, but the role of USP11 in DPC repair is not known. SPRTN is a replication-coupled DNA-dependent metalloprotease that cleaves proteins cross-linked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN auto-proteolysis and chromatin accessibility during DPC repair. Previously, VCPIP1 and USP7 deubiquitinases have been shown to regulate SPRTN. Here, we identify USP11 as an SPRTN deubiquitinase. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells and in vitro. USP11 depletion impairs SPRTN deubiquitination and promotes SPRTN auto-proteolysis in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC-inducing agents. Our findings show that USP11 regulates SPRTN auto-proteolysis and SPRTN-mediated DPC repair to maintain genome stability.

SUBMITTER: Perry M

PROVIDER: S-EPMC7960550 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Repair.

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USP11 deubiquitinates monoubiquitinated SPRTN to repair DNA-protein crosslinks

Project description:DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription, and recombination. SPRTN is a replication-coupled DNA-dependent metalloprotease that degrades proteins crosslinked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN chromatin accessibility during DPC repair. The deubiquitinase regulating SPRTN function in DPC repair is unknown. To identify SPRTN modifiers, we performed tandem-affinity purification and mass spectrometry (TAP-MS) analysis of SFB (S-FLAG-streptavidin binding peptide)-tagged SPRTN expressed in HEK 293T cells. We screened the MS list for proteins that are known to function as a deubiquitinase and identified only one potential SPRTN modifier, USP11 deubiquitinase. A reciprocal TAP-MS analysis of SFB-USP11 expressed in HEK 293T cells treated with camptothecin (CPT) immunoprecipitated SPRTN. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells and in vitro. USP11 depletion impaired SPRTN deubiquitination in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC-inducing agents. Our findings elucidates the function of USP11 in the regulation of SPRTN monoubiquitination and SPRTN-mediated DPC repair.

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Project description:Repair of covalent DNA-protein crosslinks (DPCs) by the metalloprotease SPRTN prevents genome instability, premature aging and carcinogenesis. SPRTN is specifically activated by DNA structures containing single- and double-stranded features, but degrades the protein components of DPCs promiscuously and independent of amino acid sequence. This lack of specificity is useful to target diverse protein adducts, however, it requires tight control in return, in order to prohibit uncontrolled proteolysis of chromatin proteins. Here, we discover the components and principles of a ubiquitin switch, which negatively regulates SPRTN. We demonstrate that monoubiquitylation is induced in an E3 ligase-independent manner and, in contrast to previous assumptions, does not control chromatin access of the enzyme. Data obtained in cells and in vitro reveal that monoubiquitylation induces inactivation of the enzyme by triggering autocatalytic cleavage in trans while also priming SPRTN for proteasomal degradation in cis. Finally, we show that the deubiquitylating enzyme USP7 antagonizes this negative control of SPRTN in the presence of DPCs.

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Project description:Cytotoxicity of DNA-protein crosslinks (DPCs) is ascribed largely to their ability to block the progression of DNA replication fork. DPCs are frequently occurring in cells, either as a consequence of metabolism or exogenous agents. The mechanism of DPCs removal is not completely understood. Here, we characterize SPRTN (DVC1) as specialised DNA-dependent metalloprotease for DPC removal in humans. SPRTN has an N-terminal metalloprotease domain that cleaves various DNA binding substrate during S-phase progression. SPRTN is a part of replisome and removes DPCs during DNA replication fork progression, thus protecting proliferative cells from DPCs toxicity. Ruijs-Aalfs Syndrome (RJALS) patient cells with monogenic mutations in SPRTN are hypersensitive to DPC-inducing agents due to DPC removal defect and DNA replication fork stalling. We propose a model where SPRTN protease forms specialised DNA-replication coupled DPC removal pathway essential for DNA replication fork progression and genome stability. We conclude RJALS is the first human syndrome linked to this pathway

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Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts.

Project description:DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.

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Tandem Deubiquitination and Acetylation of SPRTN Promotes DNA-Protein Crosslink Repair and Protects against Aging.

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