Replicative DNA polymerases have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, their processivity is greatly enhanced by binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. By using an integrative approach which combines cryo-electron microscopy and X-ray crystallography, the Unit of Structural Dynamics of Macromolecules deciphered the recruitment and cooperativity mechanisms between DNA polymerase D and PCNA.
DNA replication is one of the most important functions in living organisms and viruses. It ensures the integrity of the genome and the accurate transfer of genetic information. DNA polymerases (DNAPs) are the key enzymes of DNA replication and diverse DNA repair processes. Genomic DNA replication is carried out by so-called replicative DNAPs, which have evolved to copy the genome with high processivity and fidelity. Across every domain of life, polymerase holoenzyme accessory proteins play an integral role in achieving the extraordinary efficacy and accuracy of the replicative polymerase complex. These include a sliding clamp that encircles the DNA and greatly enhances the processivity. The bacterial sliding clamp is referred to as the b clamp, while the eukaryotic and archaeal sliding clamp protein is called the proliferative cell nuclear antigen (PCNA).
In eukaryotes and archaea, PCNA stimulates processive DNA synthesis of both lagging and leading strands. PCNA inhibition is therefore considered as a valuable anticancer strategy. Organisms within the archaeal domain of life possess a simplified version of the eukaryotic DNA replication machinery. PolD is an archaeal replicative DNA polymerase, which is widely distributed among Archaea and has been shown to be essential for cell viability. Similar to other replicative DNA polymerases, the activity of PolD is strongly stimulated through its interaction with PCNA. PCNA binding partners carry short motifs known as the PCNA-interacting protein-box (PIP-box).
In their work, the Unit of Structural Dynamics of Macromolecules presented the structure of the DNA-bound PolD-PCNA complex from P. abyssi at 3.77 Å using an integrative structural biology approach, combining cryo-EM, X-ray crystallography, protein-protein interaction measurements and activity assays. This structure unveils the molecular basis for the interaction and cooperativity between the whole replicative polymerase and PCNA with an unprecedented level of detail. PolD recruits PCNA via a complex mechanism, which requires two different PIP-box motifs, a C-terminal and an internal one that has never been characterized so far. They infered that the C-terminal PIP-box, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation phase and a processive phase during replication.
Source: Nat Commun 11, 27 March 2020. doi.org/10.1038/s41467-020-15392-9