Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex

Abstract

DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally switches between the polymerizing and editing modes. Here, we present the threedimensional structure of the Pyrococcus furiosus DNA polymerase B-PCNA-DNA ternary complex, which is the core component of the replisome, determined by single particle electron microscopy of negatively stained samples. This structural view, representing the complex in the editing mode, revealed the whole domain configuration of the trimeric PCNA ring and the DNA polymerase, including protein-protein and protein-DNA contacts. Notably, besides the authentic DNA polymerase-PCNA interaction through a PCNAinteracting protein (PIP) box, a novel contact was found between DNA polymerase and the PCNA subunit adjacent to that with the PIP contact. This contact appears to be responsible for the configuration of the complex specific for the editing mode. The DNA was located almost at the center of PCNA and exhibited a substantial and particular tilt angle against the PCNA ring plane. The obtained molecular architecture of the complex, including the new contact found in this work, provides clearer insights into the switching mechanism between the two distinct modes, thus highlighting the functional significance of PCNA in the replication process.fidelity control | protein-DNA complex | replication fork | single particle analysis | structural bioinformatics

The architecture of an Okazaki fragment-processing holoenzyme from the archaeon Sulfolobus solfataricus

“…The fully assembled Okazakisome (Figures 2 and 3) exhibits features compatible with previous EM studies of related subassemblies (PCNA-Lig-DNA [5] and PCNA-Pol-DNA [12]). To glean insight into the mechanism by which PCNA co-ordinates three client proteins to process Okazaki fragments, we fitted each constituent of the assembly using Chimera, based on their volumes and shapes (Figure 3).…”

“…1S5J fits extremely well in the density connecting PCNA2 and PCNA3 (Figure 3), forming two extensive contacts with the PCNA ring, consistent with the EM analysis of the PCNA-polymerase complex loaded on DNA [12].…”

“…This is analogous to what was shown in the crystallographic study of the SsoPCNA1/'2 dimer associated to Fen1 [17], where the conformation of the endonuclease recalls the Y-chain in 1UL1. We used the EM reconstructions for PCNA-LigI-DNA and PCNA-PolBI-DNA (maps EMDB-5220 and EMDB-1485 [5,12]) as probes to assign densities for PCNA-containing subcomplexes. To fit individual client proteins, we used the PDB codes: 1UL1 [16] and 2IZO [17] for the PCNA-Fen1 complex ( Figure 3B), 1S5J [18] for PolB ( Figure 3C) and 2HIV [19] for the ligase.…”

“…The PCNA-PolB1-Fen1-DNA structure was solved using the EMD-5220 map [12] as a starting model and refined with EMAN using 6878 images sampled at 6.4 Å/pixel to a resolution of ∼30 Å.…”

Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability

“…Because further extension of the DNA would sterically clash with the ring, such a large tilting angle appears unlikely in this case (41). EM analysis of PCNA/Pol-B/DNA and PCNA/ligase/DNA assemblies suggested that DNA was tilted by 13°and 16°, respectively (42,43). By contrast, in the replication factor C/ PCNA/DNA ternary assembly, the DNA axis was found to be nearly perpendicular to the PCNA ring (44).…”