Mechanism of replication machinery assembly as revealed by the DNA ligase–PCNA–DNA complex architecture

Abstract

The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.DNA replication ͉ electron microscopy ͉ single-particle analysis ͉ DNA sliding clamp ͉ protein-DNA complex D NA ligase plays essential roles in various DNA transactions, such as joining Okazaki fragments in DNA replication and nick-sealing at the final step of several DNA repair pathways, including nucleotide excision repair, base excision repair, and mismatch repair (1, 2). The enzyme catalyzes phosphodiester bond formation at the nicks generated within dsDNA, through the well-conserved 3-step reaction using either NAD ϩ or ATP as a cofactor (3). At the first step, the DNA ligase interacts with the nucleotide cofactor to form a covalent ligase-nucleoside monophosphate. At the second step, the bound AMP is transferred to the 5Ј terminus of the DNA, and finally, a phosphodiester bond is formed by a reaction between the 5Ј-DNAadenylate and the 3Ј-hydroxy group.To date, 3 crystal structures of ATP-dependent eukaryotictype DNA ligases have been reported. The structures of the 2 archaeal DNA ligases from Pyrococcus furiosus (PfuLig) and Sulfolobus solfataricus (SsoLig) were determined in the closed (4) and extended forms (5), respectively. The structure of the human DNA ligase 1 (hLigI) in complex with DNA (6) revealed that the enzyme entirely encircles the nicked DNA. All of these DNA ligases are in common composed of 3 domains, designated as the DNA binding domain (DBD), the adenylation domain (AdD), and the OB-fold domain (OBD), in the sequences from the N to C termini. Although the internal architectures of these domains are strikingly similar among the 3 DNA ligases, their relative domain orientations within each enzyme are quite different. Similar to many other replication factors, such as DNA polymerase and Flap endonuclease 1 (FEN1), DNA ligases exhibit the full activity by binding to proliferating cell nuclear antigen (PCNA). A small-angle X-ray scattering analysis revealed that the morphology of SsoLig in complex with PCNA coincides with the extended structure of SsoLig alone (5). However, the structure of the ternary Lig-PCNA-DNA complex remains unknown.PCNA interacts with various protein factors to contr...

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).…”

“…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.…”

“…It is unclear how PCNA effects hand-off of DNA from polymerase and Fen1 to DNA ligase, thereby coordinating Okazaki fragments maturation. Biochemical studies have put forward a molecular tool-belt model, although this remains controversial [3][4][5]. However, recent biochemical studies [2] support a model where a single PCNA ring assembles an Okazaki fragment maturation complex composed of PolB1, Fen1 and Lig1.…”

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).…”

Proliferating cell nuclear antigen restores the enzymatic activity of a DNA ligase I deficient in DNA binding

“…Pascal and coworkers suggested that the initial interaction of DNA ligase with PCNA occurs via its PIP box and upon PCNA binding, DNA ligase I undergoes a rearrangement to a closed‐ring conformation 16. The DNA‐binding domain (DBD) of HsDNAligI interacts with PCNA suggesting that the interaction of DNA ligase and PCNA is not dependent on the PIP box 17 and electron microscopy studies support the notion that DNA ligase and PCNA interact via a conserved protein surface 18. Despite the existence of a surface interaction between HsPCNA and HsDNAligI, the effect of this interaction on the enzymatic activity of human DNA ligase is not as clear.…”