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. 2024 Jan 25;52(2):525-547.
doi: 10.1093/nar/gkad1178.

Isolation and detection of DNA-protein crosslinks in mammalian cells

Ignacio Torrecilla  1 , Annamaria Ruggiano  1 , Kostantin Kiianitsa  2 , Ftoon Aljarbou  1 , Pauline Lascaux  1 , Gwendoline Hoslett  1 , Wei Song  1 , Nancy Maizels  2   3 , Kristijan Ramadan  1
Affiliations

Isolation and detection of DNA-protein crosslinks in mammalian cells

Ignacio Torrecilla et al. Nucleic Acids Res. .

Abstract

DNA-protein crosslinks (DPCs) are toxic DNA lesions wherein a protein is covalently attached to DNA. If not rapidly repaired, DPCs create obstacles that disturb DNA replication, transcription and DNA damage repair, ultimately leading to genome instability. The persistence of DPCs is associated with premature ageing, cancer and neurodegeneration. In mammalian cells, the repair of DPCs mainly relies on the proteolytic activities of SPRTN and the 26S proteasome, complemented by other enzymes including TDP1/2 and the MRN complex, and many of the activities involved are essential, restricting genetic approaches. For many years, the study of DPC repair in mammalian cells was hindered by the lack of standardised assays, most notably assays that reliably quantified the proteins or proteolytic fragments covalently bound to DNA. Recent interest in the field has spurred the development of several biochemical methods for DPC analysis. Here, we critically analyse the latest techniques for DPC isolation and the benefits and drawbacks of each. We aim to assist researchers in selecting the most suitable isolation method for their experimental requirements and questions, and to facilitate the comparison of results across different laboratories using different approaches.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Examples of DNA–protein crosslinks, as detected in vitro. DPCs induced by reactions with exogenous crosslinkers (in red) or by endogenous enzyme: (A) chemical structure of formaldehyde-induced DPC linking a lysine residue with N7-guanine, highlighting the resulting methylene bridge. (B) Structure of covalent linkage of TOP1 Tyr723 in the TOP1 active site to the 3′-DNA end phosphate group, highlighting the Tyr bridge in red. (C) cisplatin-induced DPC linking a lysine residue with N7-guanine, highlighting the cisplatin bridge in red. (D) 1,3-Butadiene-induced DPC on N7-guanine. (E) Direct crosslink by a free radical between tyrosine and the methyl group in thymine.
Figure 2.
Figure 2.
Replication-dependent and post-replicative DPC repair. The three main repair pathways are highlighted in pink boxes. (A) DPCs ahead of the replication fork can cause prolonged stalling and fork collapse, which is fixed by homologous recombination. Similarly, replication forks running into a TOP1cc result in single-ended DSBs which requires homologous recombination for repair. Processing of enzymatic and non-enzymatic DPCs by proteolysis is facilitated by ubiquitylation and SUMOylation. (B) SPRTN and the proteasome are the main proteolytic activities during and post-replication. The remnant peptide ahead of replication forks can be bypassed by a translesion (TLS) polymerase. Post-replicative remnant peptides are repaired by nucleases of the nucleotide excision repair (NER) pathway, which in mammalian cells can only operate on small DPCs.
Figure 3.
Figure 3.
Caesium chloride gradient method. Schematic of CsCl gradient method according to (76).
Figure 4.
Figure 4.
RADAR and STAR methods. (A) Schematic of RADAR method according to (85). (B) Schematic of STAR according to (31).
Figure 5.
Figure 5.
PxP method. Schematic of PxP method according to (33).
Figure 6.
Figure 6.
Labelling of DPCs with radioisotopic amino acids and fluorescence chemicals. Upper panel: pre-labelling method before DPC isolation according to (104). Bottom panel: post-labelling method after DPC isolation according to (44,45,84,107).
Figure 7.
Figure 7.
Comparison of protein repertoires identified by mass spectrometry. DPCs were induced by either depleting SPRTN from cells or by exposing cells to formaldehyde. Different DPC isolation methods: RADAR (32,85), PxP (33), STAR (31), cell types, MS instruments and bioinformatic analysis were used in each study. The graph shows the percentage of each protein group in the whole repertoire (bars) and the actual number of proteins found. HMG: high mobility group protein family.
Figure 8.
Figure 8.
KCl-SDS precipitation. Schematic of KCl-SDS method according to (122).
Figure 9.
Figure 9.
ARK assay. Schematic of the ARK method according to (121).
Figure 10.
Figure 10.
Modified comet assay. Schematic of the modified comet assay according to (131).
Figure 11.
Figure 11.
Detection of TOP1cc by immunofluorescence. (A) Schematic of the TOP1cc immunofluorescence protocol according to (138). (B) TOP1cc immunofluorescence of HeLa cells either untreated, treated with camptothecin (CPT; 50 nM, 1 hour), or 3 hours recovery after treatment. TOP1cc were stained with the primary antibody (Millipore Cat# MABE1084, RRID:AB_2756354) and the Alexa Fluor 555 secondary antibody. Scale bar, 10 μm. (C) Count of TOP1cc foci using Cell Profiler™. Significance determined by two-way ANOVA test. PFA: paraformaldehyde. See Supplementary material for raw data.
Figure 12.
Figure 12.
Visualisation of PARP1-DPC on DNA fibres. Schematic of the protocol as described in (152).
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