Unveiling the structural mechanisms of the Okazaki fragment processing pathway

Proliferating cell nuclear antigen (PCNA) and the proteins with which it interacts are vital in replicating genomic DNA and maintaining genomic integrity during DNA replication and repair. PCNA is the eukaryotic sliding clamp (a processivity promoting factor) that has critical roles in DNA replication and promoting tolerance of DNA damage specifically through its involvement in Okazaki fragment maturation as well as translesion synthesis (TLS) and chromatin assembly1,2. If these pathways are dysregulated, then mutations arise that can contribute to carcinogenesis. Targeting of some elements of Okazaki fragment maturation is a viable strategy to treat certain cancers3. Pol 𝛿, flap endonuclease I (FEN1) and ligase I - the proteins involved in Okazaki fragment maturation in humans - all form a variety of complexes with PCNA. To date there is limited information available on the structures involved in Okazaki fragment maturation in humans. Using cryo-EM, the structures of FEN1 and PCNA in complex with two different DNA substrates (a flap substrate and a nick substrate) have been elucidated. The structure of the ligase holoenzyme has been elucidated allowing identification of a novel binding site consisting of a PIP box in the DNA binding domain (DBD) of ligase I. The structure of the ligase - FEN1 toolbelt has also been elucidated demonstrating the presence of both ligase and FEN1 on PCNA simultaneously. These structures along with the previously published Pol 𝛿 holoenzyme and Pol 𝛿 and FEN1 toolbelt4 represent all the steps of Okazaki fragment maturation and allow a stepwise mechanism to be predicted.