A novel heterotetrameric structure of the crenarchaeal PCNA2–PCNA3 complex

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Abstract

Proliferating cell nuclear antigen (PCNA) is a key protein that orchestrates the arrangement of DNA-processing proteins on DNA during DNA metabolism. In crenarchaea, PCNA forms a heterotrimer (PCNA123) consisting of PCNA1, PCNA2, and PCNA3, while in most eukaryotes and many archaea PCNAs form a homotrimer. Interestingly, unique oligomeric PCNAs from Sulfolobus tokodaii were reported in which PCNA2 and PCNA3 form a heterotrimer without PCNA1. In this paper, we describe the crystal structure of the stoPCNA2–stoPCNA3 complex. While most DNA sliding clamps form ring-shaped structures, our crystal structure showed an elliptic ring-like heterotetrameric complex, differing from a previous reports. Furthermore, we investigated the composition and the dimension of the stoPCNA2–stoPCNA3 complex in the solution using gel-filtration column chromatography and small-angle X-ray scattering analyses, respectively. These results indicate that stoPCNA2 and stoPCNA3 form the heterotetramer in solution. Based on our heterotetrameric structure, we propose a possible biological role for the heterotetrameric complex as a Holliday junction clamp.

Introduction

DNA metabolism includes many vital processes, such as DNA replication, recombination, modification, and repair. The enzymatic machinery controlling DNA metabolism consists of a large number of cooperating DNA-processing proteins. DNA sliding clamp proteins play a central role in DNA metabolism by serving as a platform for orchestrating the arrangement of DNA-processing proteins on DNA (Moldovan et al., 2007). DNA sliding clamp proteins include the β-clamp in bacteria and proliferating cell nuclear antigen (PCNA) proteins in eukaryotes and archaea.

DNA sliding clamps are always composed of six similar domains that form a ring-shaped structure, even if the identity of amino acids sequences between them are low (Indiani and O’Donnell, 2006). In addition, DNA sliding clamps have two faces with very different electrostatics properties. The inner face around the central channel of the ring is positively charged; hence it can slide freely on DNA, and the outer face of the ring is negatively charged.

DNA-processing proteins generally bind the hydrophobic cleft of DNA sliding clamps through the PCNA interacting protein (PIP) box motif (Dalrymple et al., 2001). The cleft is located between the two domains of each monomer in PCNAs or on the C-terminal face in β-clamp. Interaction with the DNA sliding clamp affects the activity of DNA-processing enzymes (Tsurimoto, 1998, Warbrick, 2000, Vivona and Kelman, 2003). In addition, the structure of the DNA sliding clamp in complex with these enzymes may allow binding of more than one protein at the same time (Gulbis et al., 1996, Bruning and Shamoo, 2004, Kontopidis et al., 2005, Sakurai et al., 2005, Doré et al., 2006, Xing et al., 2009, Hishiki et al., 2009), though the mechanism of this cooperative interaction has attracted attention as an interesting topic. Furthermore, it has been reported that ubiquitination or SUMO modification of PCNA is needed to switch polymerases during the DNA-damage response (Hoege et al., 2002, Stelter and Ulrich, 2003, Pfander et al., 2005, Moldovan et al., 2006).

In bacteria, β-clamp protein forms a homodimer, while in eukaryotes or euryarchaea PCNAs form homotrimers. However, a PCNA-forming heterotrimer (PCNA123), consisting of PCNA1, PCNA2 and PCNA3, has been found in three crenarchaeal species: Sulfolobus solfataricus (ssoPCNA) (Dionne et al., 2003), Aeropyrum pernix (apePCNA) (Imamura et al., 2007), and Sulfolobus tokodaii (stoPCNA) (Lu et al., 2008). The PCNA123 heterotrimer plays a similar role to eukaryotic PCNA in that it affects the activities of DNA polymerase (PolB1), DNA Ligase I, and flap endonuclease I (Fen-I) (Dionne et al., 2003). The loading mechanisms of both PCNA123 and eukaryotic PCNA are also similar (Indiani and O’Donnell, 2006, Dionne et al., 2008).

Biochemical studies of PCNA1, PCNA2, and PCNA3 heterotrimer formation indicate that the subunit interactions vary depending on the species. In S. solfataricus for example, ssoPCNA1 and ssoPCNA2 first form a stable heterodimer and then it recruits ssoPCNA3. In S. tokodaii or A. pernix, stoPCNA3 or apePCNA2 interacts with respective other subunits, while no interaction (or little affinity) is observed between the others (Dionne et al., 2003). Moreover, PCNA2 and PCNA3 were reported to form a heterotrimer that does not include PCNA1 in S. tokodaii (Lu et al., 2008) and A. pernix (Imamura et al., 2007). The A. pernix subunit (apePCNA2) forms a homo-oligomeric complex that is roughly the size of the apePCNA123 and apePCNA2–apePCNA3 complexes. The stoPCNA2–stoPCNA3 complex enhances by twofold the enzyme activity of the Holliday junction resolvase Hjc compared to stoPCNA123, while the stoPCNA2–stoPCNA3 complex reduces the enzymatic activity of the RecQ-like helicase Hjm and DNA Ligase I similarly to stoPCNA123 (Lu et al., 2008). In addition, the apePCNA2–apePCNA3 complex enhances the enzymatic activity of DNA polymerase, DNA Ligase I, and Fen-1 similarly to apePCNA123 (Imamura et al., 2007). Although the crystal structures of PCNA123 have been determined (Williams et al., 2006, Hlinkova et al., 2008), the structure of PCNA2–PCNA3 complex is still unknown.

In this study, we determined the crystal structure of the stoPCNA2–stoPCNA3 complex to reveal the structural differences that exist between the PCNA2–PCNA3 complex and PCNA123. It has been reported that the stoPCNA2–stoPCNA3 complex forms a heterotrimer consisting of one stoPCNA2 molecule and two stoPCNA3 molecules (Lu et al., 2008). However, our present study reveals that the stoPCNA2–stoPCNA3 complex forms a heterotetramer in nature, consisting of two molecules each of stoPCNA2 and stoPCNA3. The heterotetrameric structure of the stoPCNA2–stoPCNA3 complex in the aqueous solution was further validated by gel filtration chromatography and small-angle X-ray scattering (SAXS). Based on this new information regarding its structural features, we hypothesize a possible biological role of stoPCNA2–stoPCNA3 complex as a Holliday junction clamp similar to RuvA.

Section snippets

Expression, purification, and crystallization of stoPCNAs

Crystals of the stoPCNA2–stoPCNA3 complex were prepared as described previously (Kawai et al., 2009). Briefly, stoPCNA monomers were individually overexpressed in E. coli strain BL21(DE3) using the pET expression system (Novagen). Proteins were purified by heat treatment, ammonium sulfate precipitation, and HiTrap Q (GE Healthcare) anion-exchange chromatography. The stoPCNA2–stoPCNA3 complex was formed by mixing the respective stoPCNA monomers in an equal molar ratio and the complex was

Crystal structure of the stoPCNA2–stoPCNA3 complex

The stoPCNA2–stoPCNA3 complex crystallized in space group I222 with two molecules (one molecule each of stoPCNA2 and stoPCNA3) per asymmetric unit, and crystallized in space group P21212 with four molecules (two molecules each of stoPCNA2 and stoPCNA3) per asymmetric unit. Data-collection and structure refinement statistics are summarized in Table 1. In the crystal, stoPCNA2 and stoPCNA3 form an elliptic ring-like heterotetramer consisting of two stoPCNA2 and two stoPCNA3 molecules in which

The stoPCNA2–stoPCNA3 complex as a heterotetramer

Our X-ray crystallographic study is the first characterization of the novel heterotetrameric structure of the stoPCNA2–stoPCNA3 complex. Gel filtration chromatography followed by SDS–PAGE analysis and SAXS also indicated that stoPCNA2 and stoPCNA3 form a heterotetramer in the aqueous solution. Previous study based on the results of pull-down assays using His-tag or GFP fusion proteins have shown that the stoPCNA2–stoPCNA3 complex exists a heterotrimer rather than heterotetramer (Lu et al., 2008

Acknowledgments

We are grateful to Dr. Eiki Yamashita, Professor Atsushi Nakagawa, and the beamline staff for their support at the BL44XU in the SPring-8. Synchrotron experiments were performed with the approval of the Joint Research Committee of the Institute for Protein Research, Osaka University, and the Japan Synchrotron Radiation Research Institute (Proposal No. 2008B6831). This work was partly supported by the National Project on Protein Structural and Functional Analysis by the Ministry of Education,

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