Larsen NB et al. (2019), Replication-Coupled DNA-Protein Crosslink Repai...

XB-ART-55583

Mol Cell 2019 Feb 07;733:574-588.e7. doi: 10.1016/j.molcel.2018.11.024.

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

Larsen NB , Gao AO , Sparks JL , Gallina I , Wu RA , Mann M , Räschle M , Walter JC , Duxin JP .

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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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Species referenced: Xenopus laevis
Genes referenced: cstb gem gins4 gmnn mcm6.2 orc2 rev1 sprtn

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	Figure 1. Replication-Coupled Ubiquitylation and Degradation of a DPC(A) Schematic of the DPC recovery assay.(B) pDPC was replicated in egg extracts. Geminin (+ Gem.) was added where indicated to block DNA replication. DPCs were recovered as illustrated in (A) at the indicated time points, and DPCs were blotted with a M.HpaII antibody. Input samples were blotted with an origin recognition complex subunit 2 (ORC2) antibody.(C) pDPC2xLead, a plasmid containing two DPCs (one on each leading strand; see Figure 2A), was replicated in egg extracts supplemented with free ubiquitin (Ub) or FLAG-ubiquitin. At 20 min, DPCs were recovered and blotted as in (B). Red arrowheads indicate the mobility shift induced by FLAG-ubiquitin.(D) pDPC2xLead was replicated in egg extracts supplemented with free ubiquitin (Ub) or FLAG-ubiquitin. At the indicated time point, DPCs were recovered as in (B) (Input) and immunoprecipitated with anti-FLAG-resin (FLAG-IP). Ubiquitylated DPCs were detected with M.HpaII antibody. Red arrowheads indicate the location of mono-, di-, and tri-ubiquitylated M.HpaII.(E) pDPCLead or pDPCLag was replicated in egg extract in the presence of LacI to ensure that a single replication fork encounters the DPC (Duxin et al., 2014). Recovered DPCs were blotted as in (B). The asterisk indicates residual uncrosslinked M.HpaII.
	Figure 2. SPRTN and the Proteasome Are Recruited to a DPC Plasmid during Replication(A) Depiction of replication, recovery, and analysis of pDPC2xLead by PP-MS. To monitor the progress of the repair reaction, pDPC2xLead was replicated in the presence of [α-32P]dATP, and replication intermediates were analyzed by agarose gel electrophoresis (lower autoradiograph). In parallel, plasmids were isolated together with the bound proteins by a LacI pull-down (Budzowska et al., 2015) and analyzed by label-free MS.(B) Heatmap showing the mean of the Z scored log2 label-free quantitation LFQ intensity from four biochemical replicates of pCTRL and pDPC2xLead. Geminin was added to block replication where indicated.(C) Analysis of protein recruitment to pDPC2xLead compared to pCTRL. Both plasmids were recovered at 40 min. The volcano plot shows the mean difference of the protein intensity plotted against the p value calculated by a modified, one-sided t test. Full results are reported in Tables S1 and S2.
	Figure 3. SPRTN and the Proteasome Degrade DPCs during Replication(A) Mock-depleted and SPRTN-depleted egg extracts were blotted with SPRTN and MCM6 (loading control) antibodies.(B) The extracts from (A) were used to replicate pDPC2xLead in the presence of [α-32P]dATP. MG262 at 200 μM was added where indicated. Samples were analyzed by native agarose gel electrophoresis. Red arrowheads indicate the accumulation of OC repair intermediates. Note that OC molecules that accumulate are subjected to 5′ to 3′ end resection and smear-down on the gel (lanes 14, 19, and 20;Duxin et al., 2014). Replication intermediates (RI), open circular (OC), and supercoiled species (SC) were quantified as a percentage of total lane signal (lower graphs). The mean percentages across three independent experiments are plotted, with error bars representing the SD.(C) DPCs from (B) were recovered and monitored as in Figure 1B.(D) Mock-depleted and SPRTN-depleted egg extracts were blotted with SPRTN and MCM6 (loading control) antibodies. SPRTN-depleted extracts were supplemented with either buffer (+Buf), recombinant FLAG-SPRTN (+WT), or recombinant catalytically inactive FLAG-SPRTN E89Q (+EQ).(E) The extracts from (D) were used to replicate pDPC2xLead in the presence of [α-32P]dATP. MG262 at 200 μM was added where indicated. Samples were analyzed and quantified as in (B). The quantification of a representative biological replicate is shown.(F) DPCs from (E) were monitored as in Figure 1B.(G) Mock-depleted, SPRTN-depleted, or proteasome subunit α type-1 (PSMA1)-depleted extracts were used to replicate pDPC2xLead. Plasmids were recovered, and protein-recruitment to the plasmid was monitored with the indicated antibodies (Budzowska et al., 2015).
	Figure 4. SPRTN, but Not the Proteasome, Can Degrade Non-ubiquitylated DPCs(A) Strategy to address the role of DPC ubiquitylation via reductive methylation of the DPC.(B) pDPC2xLead and pme-DPC2xLead were replicated in egg extracts, and DPCs were monitored as in Figure 1B. Note the concomitant disappearance of full-length M.HpaII and appearance of a degradation product during replication of pme-DPC2xLead. Both a long and a short exposure of the M.HpaII blot are shown.(C) pDPC2xLead and pme-DPC2xLead were replicated in egg extracts, and recruitment of the indicated proteins to the plasmid was monitored as in Figure 3G.(D) pme-DPC2xLead was replicated in mock-depleted or SPRTN-depleted egg extracts. DPCs were monitored as in Figure 1B.(E) pme-DPC2xLead was replicated in mock-depleted and SPRTN-depleted extracts. SPRTN-depleted extracts were supplemented with either buffer (+buf), or recombinant FLAG-SPRTN variants (see Figure S4D). DPCs were monitored as in Figure 1B.(F) pDPC2xLead and pme-DPC2xLead were replicated in mock-depleted or SPRTN-depleted extracts. Samples were analyzed as in Figure 3B. The mean of three independent experiments is quantified. Error bars represent the SD.(G) Samples from (F) were digested with FspI and AatII and separated on a denaturing polyacrylamide gel. The schematic depicts the nascent leading strands and extension products liberated by FspI and AatII digestion (green hexamer, CMG helicase; red lines, nascent DNA). The locations of the corresponding bands on the gel are indicated by brackets. The −30 to −40 species, the −1,0,1 species, and extension products were quantified and plotted below. Quantification of each species is plotted as a percentage of the entire signal of the lane. The quantification of a representative biological replicate is shown.
	Figure 5. DPC Ubiquitylation and Degradation Can Occur in the Absence of the Replisome(A) Schematic comparing pDPC and pDPCssDNA in non-licensing egg extracts.(B) pDPC and pDPCssDNA were incubated in non-licensing egg extracts. DPCs were recovered and monitored as in Figure 1B. Note that time 0 was withdrawn before incubating plasmids in egg extracts, which explains the absence of ORC2 input in lanes 1 and 5.(C) pDPCssDNA was incubated in mock-depleted and SPRTN-depleted non-licensing extracts in the presence of 200 μM MG262 where indicated. DPCs were monitored as in Figure 1B.(D) Schematic comparing the fate of pDPCssDNA in the presence or absence of gap-filling synthesis.(E) pDPCssDNA was incubated in non-licensing egg extracts in the presence of [α-32P]dATP. Extracts were supplemented with 700 μM aphidicolin and 1 mM araCTP where indicated. Samples were analyzed as in Figure 3B.(F) Samples from (E) were digested with PvuII and NdeI and separated on a denaturing polyacrylamide gel. The different extension products are depicted in the upper scheme.(G) Samples from (E) were used to monitor DPC ubiquitylation and degradation as in Figure 1B.
	Figure 6. SPRTN-Dependent DPC Degradation Requires Nascent Strand Extension to the Lesion(A) Schematic comparing the fate of pme-DPCssDNA in the presence and absence of gap-filling synthesis.(B) pme-DPCssDNA was incubated in non-licensing egg extracts supplemented with 700 μM aphidicolin and 1 mM araCTP where indicated. DPC degradation was monitored as in Figure 1B. The asterisk denotes a crosslinked methyl-M.HpaII species generated on the GAP substrate, likely caused by the incomplete degradation of ssDNA by benzonase.(C) Samples from (B) were analyzed as in Figure 5F.(D) Depiction of pme-DPC+peptide replication.(E) pme-DPC and pme-DPC+peptide were replicated in REV1-depleted extracts in the presence of [α-32P]dATP. Samples were digested with Nb.BsmI, which cuts the leftward leading strand, as depicted in (D). Nascent leading strands were then separated on a polyacrylamide denaturing gel.(F) Samples from (E) were used to monitor DPC degradation as in Figure 1B.
	Figure 7. TRAIP Ubiquitin Ligase Stimulates DPC Ubiquitylation and Proteasome Targeting(A) Heatmap showing the mean of the Z scored log2 LFQ intensity of potential E3 ubiquitin ligases. Proteins with similar intensities in the geminin or mock control lacking the DNA substrate were excluded.(B) Extracts were depleted with SPRTN and either control immunoglobulin G (IgG) or TRAIP antibodies and blotted for TRAIP and RTEL1 (loading control). TRAIP-depleted extracts were supplemented with buffer (+Buf), recombinant TRAIP(WT), or TRAIP(R18C).(C) Extracts from (B) were used to replicate pDPC2xLead. DPCs were monitored as in Figure 1B.(D) SPRTN- and RTEL1-depleted extracts were either mock depleted or TRAIP depleted. TRAIP-depleted extracts were supplemented with buffer (+Buf), recombinant TRAIP(WT), or TRAIP(R18C). These extracts were used to replicate pDPC2xLead, and DPCs were monitored as in Figure 1B.(E) The indicated extracts were used to replicate pDPC2xLead. Recruitment of the indicated proteins to the plasmid was monitored as in Figure 3G.(F) Extracts described in (D) were used to replicate pDPC2xLead in the presence of [α-32P]dATP, and nascent strand intermediates were analyzed as in Figure 4G. CMG bypass was measured based on the disappearance of the −30 to −40 CMG footprint (Sparks et al., 2018). The mean of three independent experiments is graphed for mock-, TRAIP-, and RTEL1-TRAIP-depleted samples. Error bars represent the SD. The TRAIP-RTEL1-depleted samples supplemented with TRAIP(WT) represents the mean of two experiments and plotted without error bars.(G) Samples from (F) were blotted with TRAIP, RTEL1, or SLD5 (loading control) antibodies.(H) Model for replication-coupled DPC proteolysis in Xenopus egg extracts. Black lines, parental DNA; red lines, nascent DNA; green hexamers, CMG helicase; blue spheres, replicative polymerases; yellow spheres, TLS polymerase; gray sphere, DPC; orange, SPRTN; yellow and blue, the proteasome; dark green, TRAIP-dependent ubiquitin chains; light green, ubiquitin chains deposited by a second E3 ligase activated by ssDNA.

References [+] :

Azuma, SUMO-2/3 regulates topoisomerase II in mitosis. 2003, Pubmed, Xenbase