Cryo-EM structure and dynamics of eukaryotic DNA polymerase δ holoenzyme

Abstract

DNA polymerase δ (Polδ) plays pivotal roles in eukaryotic DNA replication and repair. Polδ is conserved from yeast to humans, and mutations in human Polδ have been implicated in various cancers. Saccharomyces cerevisiae Polδ consists of catalytic Pol3 and the regulatory Pol31 and Pol32 subunits. Here, we present the near atomic resolution (3.2 Å) cryo-EM structure of yeast Polδ holoenzyme in the act of DNA synthesis. The structure reveals an unexpected arrangement in which the regulatory subunits (Pol31 and Pol32) lie next to the exonuclease domain of Pol3 but do not engage the DNA. The Pol3 C-terminal domain contains a 4Fe-4S cluster and emerges as the keystone of Polδ assembly. We also show that the catalytic and regulatory subunits rotate relative to each other and that this is an intrinsic feature of the Polδ architecture. Collectively, the structure provides a framework for understanding DNA transactions at the replication fork.

Fig. 1 |. Cryo-EM structure of DNA bound Polδ holoenzyme.

a, Schematic of the primary (left) and quaternary (right) structure of S. cerevisiae Polδ. b,c, Cryo-EM density map of Polδ colored by local resolution (b) and domains (c). d, Polδ structure fitted into the cryo-EM map. e, Structure of Polδ colored by domain. The region of the template-primer duplex enclosed in the box was built into the final model.

Fig. 2 |. Structure and cryo-EM density for selected regions of catalytic and regulatory modules and CysBD.

a, Well resolved density for the pol and exo active sites, including ligands, metal ions and water. Active sites in Polδ are configured as in the isolated catalytic module. (PDB 3IAY). b, Clear density for the regulatory module enabled modeling of regions that are not resolved in the human homolog (PDB 3E0J). c, Ab initio structure of 4Fe-4S cluster bound CysBD. Some side chains have been omitted for clarity. d, Sequence alignment of a section of CysBD spanning the conserved Arg1080.

Fig. 3 |. CysBD is the keystone of Polδ assembly.

a, Simultaneous interactions of CysBD with the regulatory and catalytic modules (in yellow and red respectively and dotted surface) nucleates the assembly of Polδ. CysBD amino acids tested previously in genetic experiments for interactions and those identified from our structure are labeled. b, Potential contacts between the regulatory and catalytic modules are mapped on the schematic and listed in the legend below. c, Human Polδ disease mutations R506H, R507C and I1070N corresponding to R511, R512 and L1068 mapped on a schematic of the structure of yeast Polδ. These mutations localize at the interface between the catalytic and regulatory modules.

Fig. 4 |. Flexibility between catalytic and regulatory modules.

a, Comparison of consensus and multi-body maps for selected regions of Pol31 and Pol32. Multi-body refinement results in significant improvement in the resolution of Pol32N and regions of Pol31 that are far from the catalytic module. b,c, Motion represented by the first and second eigenvectors from multi-body analysis. The first vector represents a rocking motion parallel to the exo domain of the catalytic module, while the second vector represents a rocking motion towards the exo domain.