Mechanisms of DNA repair and DNA replication in the Duxin Group

DPCs are common DNA lesions generated by a wide variety of crosslinking agents. For example, many chemotherapeutic drugs used in the clinic cause DPCs. Failure to repair DPCs interfere with DNA metabolism and threatens genomic integrity causing accelerated ageing and cancer in humans.

 “Understanding DPC repair is critical for human health, as many chemotherapeutic drugs used in the clinic exert their toxicity by generating DPCs and we know very little about the processes involved in clearing these lesions from the genome,” says Associate Professor and Group Leader Julien Duxin.

To this end, the Duxin group takes advantage of a protein extract system derived from eggs from African clawed frogs (Xenopus laevis). These protein extracts have the remarkable capacity to reiterate processes of DNA replication and DNA repair in a cell-free environment, providing a unique opportunity to delineate the molecular mechanisms underlying complex DNA transactions.  

“Using frog egg extracts we are able to recapitulate DPC-repair in a test tube. This allows us to manipulate this process and interrogate which are the main players orchestrating this reaction,“ says Julien Duxin.

New Role for ubiquitin ligase RFWD3
Using Xenopus egg extracts the Duxin group identified a new role for the ubiquitin ligase RFWD3. They found that RFWD3 promotes ubiquitylation of PCNA and proteins on single-stranded DNA to stimulate gap filling DNA synthesis across different polymerase-blocking DNA lesions including DNA-protein and DNA-interstrand crosslinks. This provides a molecular understanding of RFWD3’s essential function in safeguarding the genome.
"The ubiquitin ligase RFWD3 is required for translesion DNA synthesis"  
Gallina et al., Molecular Cell, 2021

Mechanisms of replication-coupled DPC proteolysis
A new study from the Duxin group presents two mechanisms of replication-coupled DPC proteolysis and provide the molecular links that couple these processes to DNA replication. By employing different DPC proteases, cells possess a versatile system that can cope with a wide diversity of DPCs.
“Replication-Coupled DNA-Protein Crosslink Repair by SPRTN and the Proteasome in Xenopus Egg Extracts"
Larsen et al., Molecular Cell, 2019

DPC repair mechanism
During his post-doctoral training, Dr Julien Duxin was able to recapitulate DNA-protein crosslink repair using Xenopus egg extracts. This study revealed how this repair process is tightly coupled to DNA replication.
“Repair of a DNA-protein crosslink by replication-coupled proteolysis” 
Duxin et al., Cell, 2014