Book of Abstracts: Albany 2007
June 19-23 2007
Crystallographic and Small-angle X-ray Scattering Analysis of DNA Ligase Interaction with the Protein Sliding Clamp, PCNA
Many enzymatic activities are coordinated at the DNA replication fork to ensure an accurate and efficient duplication of the genome. Proliferating cell nuclear antigen (PCNA) is a trimeric, ring-shaped protein that encircles duplex DNA and serves as a docking site at the replication fork for several nucleic acid enzymes, including polymerase, endonuclease, and ligase. How PCNA coordinates the activities of its many binding partners remains unclear. We have studied the mechanism of the DNA ligase-PCNA interaction with a structural and biochemical approach using proteins from Sulfolobus solfataricus. PCNA binds DNA ligase and strongly stimulates DNA joining activity in this hyperthermophilic organism. The crystal structure of the ATP-dependent S. solfataricus DNA ligase (ssLig) shows the three-domain enzyme in an extended conformation. Small-angle x-ray scattering (SAXS) analysis of ssLig in solution also indicates that the enzyme exists in an extended, rod-like conformation. In contrast, the ligase domains are in a ring-shaped conformation in the crystal structure of the homologous human DNA ligase I bound to DNA. Therefore, a large change in ligase conformation is expected upon DNA binding. An additional active conformation of ssLig is inferred from crystal packing interactions and a complex with ATP cofactor, further highlighting the flexibility and remarkable mobility of the ligase domains during the course of the ligation reaction. We investigated whether the PCNA ring has an influence on the conformational state of DNA ligase, perhaps templating the ring-shaped conformation of ligase. Unlike the homotrimeric PCNA in humans, S. solfataricus PCNA (ssPCNA) is a heterotrimer, with each subunit providing a unique docking site for binding proteins. Our crystal structure of ssPCNA suggests a mechanism for organizing specific interactions with partner proteins on each subunit, allowing three different proteins to bind to the ssPCNA ring simultaneously in an ordered manner. SAXS analysis of the ligase-PCNA complex demonstrates that ssLig binds to one location on the outside edge of the ssPCNA ring in an extended conformation. A model of the ligase-PCNA complex based on our SAXS analysis, atomic resolution structures, and biochemical data supports three main conclusions: ssPNCA provides a docking site for ssLig without affecting the extended conformation of ssLig in the absence of DNA, ssLig domains are free to react with ATP while bound to PCNA, and the ligase-PCNA interaction is adaptable to allow ssLig to adopt the ring-shaped conformation upon encountering a DNA strand break. Future studies will focus on the dynamics of the protein interface to understand the mechanisms that allow the ligase-PCNA complex to efficiently process breaks in DNA.
John M. Pascal1
1Department of Biochemistry and Molecular Biology