Structures of the human leading strand Polε-PCNA holoenzyme

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Eukaryotic DNA replication relies on replicative polymerases, including Polε and Polδ, which exhibit enhanced processivity when associated with the proliferating cell nuclear antigen (PCNA) clamp.
• While the catalytic domain structure of yeast Polε is known, the molecular mechanism of its interaction with PCNA in any eukaryote remains unelucidated.

Purpose of the Study:

• To determine the structural basis of the interaction between human DNA polymerase epsilon (Polε) and the PCNA clamp.
• To elucidate the mechanism by which this interaction regulates DNA synthesis and nucleotide binding.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was employed to determine the structures of the human Polε-PCNA-DNA complex in two distinct functional states: incoming nucleotide bound and nucleotide exchange.
• Detailed structural analysis of the complex to identify key interaction interfaces and conformational changes.

Main Results:

• Two cryo-EM structures revealed a unique three-point interface between the Polε catalytic domain and the PCNA trimer.
• This interface involves the PIP-motif, the P-domain, and the thumb domain of Polε, each engaging a different PCNA protomer.
• Structural comparison highlighted a conformational change involving the finger domain pivoting around the P-domain's [4Fe-4S] cluster, regulating nucleotide binding and exchange.

Conclusions:

• The multi-point interaction between Polε and PCNA is crucial for stable complex formation and may prevent the recruitment of other PCNA-interacting proteins during replication.
• The observed conformational dynamics provide mechanistic insights into how Polε regulates DNA synthesis progression and nucleotide incorporation.