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

Abstract

Summary 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.

SUMO ylation promotes protective responses to DNA ‐protein crosslinks

“…Available evidence suggests that ubiquitin‐regulated SPRTN recruitment to and processing of DPCs is tightly coupled to DNA replication (Lessel et al , ; Lopez‐Mosqueda et al , ; Stingele et al , ; Vaz et al , ). In addition, recent studies using Xenopus egg extracts revealed a parallel role of the proteasome and replication fork‐associated E3 ubiquitin ligase activity in DNA replication‐coupled DPC repair (Larsen et al , ). By contrast, our findings show that chromatin SUMOylation elicited in response to DPCs does not require ongoing DNA replication but is operational throughout interphase.…”

The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast

“…Wss1 has a SUMO-interacting motif and is proposed to be regulated by HMW-SUMO species (Balakirev et al, 2015;Stingele et al, 2014). SPRTN, in contrast, possesses ubiquitin-binding motifs, binds PCNA, is cell-cycle regulated and activated by de-ubiquitination and ssDNA (Larsen et al, 2018;Mosbech et al, 2012;Stingele et al, 2016). In addition to the dedicated proteolytic mechanism, it was proposed previously and confirmed recently that the 26S proteasome is directly involved in DPC proteolysis (Klages-Mundt and Li, 2017;Larsen et al, 2018;Lin et al, 2008;Quievryn and Zhitkovich, 2000).…”

“…SPRTN, in contrast, possesses ubiquitin-binding motifs, binds PCNA, is cell-cycle regulated and activated by de-ubiquitination and ssDNA (Larsen et al, 2018;Mosbech et al, 2012;Stingele et al, 2016). In addition to the dedicated proteolytic mechanism, it was proposed previously and confirmed recently that the 26S proteasome is directly involved in DPC proteolysis (Klages-Mundt and Li, 2017;Larsen et al, 2018;Lin et al, 2008;Quievryn and Zhitkovich, 2000). In addition, the CMG helicase can bypass DPCs with the help of RTEL1 prior to proteolysis, however SPRTN and 26S proteasome activities remain the prerequisite for the full performance of a downstream repair pathway (Sparks et al, 2019).…”

The yeast proteases Ddi1 and Wss1 are both involved in the DNA replication stress response

“…As many replicationhindering barriers contain a protein component, it is unsurprising that proteases are involved in the cellular response to replication stress. The ubiquitin proteasome system plays a central role, with 26S proteasome involvement in regulated degradation of signaling molecules (Zhang et al 2005), stalled RNA polymerases (Woudstra et al 2002), and Ku70/80 proteins trapped after successful non-homologous end joining (van den Boom et al 2016), as well as in general degradation of proteins trapped on the DNA strand (Larsen et al 2018).…”