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Identification of formaldehyde-induced DNA-protein crosslinks by PxP-MS

ABSTRACT: Compromised enzymatic actions, reactive metabolites, and various chemotherapeutics cause toxic covalent DNA-protein crosslinks (DPCs). Failure to repair DPCs results in genomic instability, premature aging, and tumorigenesis. However, understanding the principles underlying DPC formation and repair has been hampered by a lack of methodologies to study DPCs in human cells. Here, we developed a technique for the Purification of x-linked Proteins (PxP), which allows identification and tracking of enzymatic and nonenzymatic DPCs. By combining PxP with quantitative proteomics, we investigated the nature of DPCs induced by formaldehyde.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ignasi Forne

LAB HEAD: Prof. Dr. Julian Stingele

PROVIDER: PXD026654 | Pride | 2023-01-20

REPOSITORIES: Pride

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			Action	DRS
	Groups.txt	Txt
	Ref1676_JS_11_20180727.raw	Raw
	Ref1676_JS_12_20180727.raw	Raw
	Ref1676_JS_13_20180727.raw	Raw
	Ref1676_JS_14_20180727.raw	Raw

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Transcription-coupled DNA-Protein Crosslink repair by CSB and CRL4(CSA)-mediated degradation

Project description:DNA-protein crosslinks (DPCs), arise from enzymatic intermediates, endogenous metabolism or exogenous chemicals like chemotherapeutics. DPCs are highly cytotoxic as they will impede DNA transacting processes such as replication, which is counteracted by proteolysis-mediated DPC removal by the protease SPRTN or the proteasome. However, how DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here, we show that DPCs severely inhibit RNA polymerase II (Pol II)-mediated transcription, resulting in the recruitment of the transcription-coupled nucleotide excision repair (TC-NER) factors CSA and CSB. Interestingly, CSA and CSB are indispensable for transcription-coupled DPC (TC-DPC) repair, while the downstream TC-NER factors UVSSA and XPA are not, indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independent of SPRTN, but is mediated by the activities of the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs are preferentially repaired in genes by a dedicated transcription-coupled repair mechanism, which is crucial to warrant correct transcription.

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CSB promotes transcription-coupled repair of DNA-protein crosslinks

Project description:PxP-MS (Purification of x-linked Proteins coupled to Mass Spectrometry) was used to assess the role of CSB in DNA-protein crosslink repair. The CSB protein is a sensor that can detect stalled RNA polymerases at sites of DNA damage, thereby triggering transcription-coupled repair mechanisms. DPCs were induced in WT and CSB knock-out RPE1 cells using a pulse of formaldehyde. To identify crosslinked proteins that specifically require CSB for repair, DNA-protein crosslinks were isolated from cells using PxP either directly after formaldehyde exposure or following a chase in drug-free media and identified by mass spectrometry.

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A SUMO-mediated protective response to DNA-protein crosslinks

Project description:DNA-protein crosslinks (DPCs) are highly cytotoxic lesions that obstruct essential DNA transactions and whose resolution is critical for cell and organismal fitness. However, unlike the repair pathways for most types of DNA damage, the mechanisms by which cells respond to and overcome DPCs remain poorly understood. Recent studies unveiled a dedicated DPC repair pathway in higher eukaryotes involving the SprT-type metalloprotease SPRTN/DVC1, which proteolytically processes DPCs during DNA replication in a ubiquitin-controlled manner. Here, we show that aldehyde-generated DPCs elicit a potent and highly dynamic, cell cycle-independent SUMOylation response impacting numerous chromatin-associated human proteins. We identify an uncharacterized SprT metalloprotease, ACRC, which unlike SPRTN is targeted to DPCs in a SUMO-dependent manner. Moreover, we establish important physiological roles of the C. elegans ACRC orthologue GCNA-1 and SUMO-mediated signaling in promoting organismal survival upon DPC formation. Finally, we demonstrate that like aldehydes, defined enzymatic DNMT1 DPCs trigger a robust SUMO response targeting the crosslinked proteins and associated factors to promote efficient lesion processing and cellular fitness. Collectively, our findings reveal an evolutionarily conserved general role of SUMO-mediated signaling in facilitating responses to DPCs in metazoans.

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- The STAR assay: A new sensitive, selective, and versatile method for isolation of DNA-protein crosslinks

Project description:- DNA-protein crosslinks (DPCs), formed by covalent conjugation of proteins to DNA molecule, are toxic DNA lesions created by exposure to physical or chemical insults or faulty enzymatic reactions. Because of their bulky nature and chemical changes to DNA molecule, they interfere with DNA metabolic processing and transcription. With the recent discovery of proteases and nucleases that participate in the repair of such lesions, the development of an accurate biochemical assay for DPC isolation became a priority for further mechanistic understanding of their repair. Here we propose the STAR assay, a novel sensitive, selective, and versatile method for the direct quantification of DPCs, with high sensitivity to physiologically relevant treatment conditions. The assay is efficient in detecting physically, chemically, or enzymatically created adducts. We demonstrate that nuclear localization is required for crosslinking the proteins to DNA molecule. Combining the STAR assay results with Comet assay and western blot confirmed that the repair of DPCs is a two-step process, starting with the removal of protein part of the lesions followed by the repair of the DNA molecule itself. The STAR assay is applicable for studying formation, repair, and biological significance of DPCs, as well as for chemotherapeutics dose management in personalized medicine.

2023-01-17 | PXD032763 | Pride

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Project description:DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors.

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