Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA - Analytical Methods & Tools for Lyophilization

Circadian clock protein KaiC forms ATP-dependent hexameric rings and binds DNA
Published online before print December 11, 2002
Tetsuya Mori , Sergei V. Saveliev , Yao Xu , Walter F. Stafford , ?, Michael M. Cox , Ross B. Inman , and Carl H. Johnson ||
PNAS | December 24, 2002 | vol. 99 | no. 26
Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235; Department of Biochemistry, University of Wisconsin, Madison, WI 53706; Boston Biomedical Research Institute, Watertown, MA 02742; and ?Department of Neurology, Harvard Medical School, Boston, MA 02115
Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved October 28, 2002 (received for review September 24, 2002)
Abstract
KaiC from Synechococcus elongatus PCC 7942 (KaiC) is an essential circadian clock protein in cyanobacteria. Previous sequence analyses suggested its inclusion in the RecA/DnaB superfamily. A characteristic of the proteins of this superfamily is that they form homohexameric complexes that bind DNA. We show here that KaiC also forms ring complexes with a central pore that can be visualized by electron microscopy. A combination of analytical ultracentrifugation and chromatographic analyses demonstrates that these complexes are hexameric. The association of KaiC molecules into hexamers depends on the presence of ATP. The KaiC sequence does not include the obvious DNA-binding motifs found in RecA or DnaB. Nevertheless, KaiC binds forked DNA substrates. These data support the inclusion of KaiC into the RecA/DnaB superfamily and have important implications for enzymatic activity of KaiC in the circadian clock mechanism that regulates global changes in gene expression patterns.
DnaB | RecA | cyanobacteria | Synechococcus
Abbreviations: KaiC, full-length KaiC from Synechococcus elongatus PCC 7942; KaiCI, the first "half" of KaiC from S. elongatus PCC 7942; KaiC-P2, full-length KaiC from the thermophilic cyanobacterium Synechococcus lividus; EM, electron microscope or electron microscopy; EMSA, electrophoretic mobility-shift assay; Rs, Stokes radius; AMP-PNP, adenosine 5'-(,-imido)-triphosphate
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Circadian rhythms are endogenous biological programs that "free-run" with a period close to 24 h in constant conditions but will entrain to appropriate environmental cycles of light/dark or temperature. Before 1985, it was believed that circadian programs were exclusively found among eukaryotes; however, it is now clearly documented that eubacterial cyanobacteria exhibit circadian rhythms (1, 2). In cyanobacteria, this circadian clockwork orchestrates rhythmic changes in the expression of nearly every gene in the organism (1, 3). A mutational analysis in the genetically tractable cyanobacterium, Synechococcus elongatus PCC 7942, pinpointed a cluster of three genes, kaiA, kaiB, and kaiC, that were essential circadian clock genes but were not required for viability (4, 5). The kaiC gene is the largest of the three, and most of the mutations that have been isolated by mutational screening were mapped to kaiC.
On the basis of a genomic survey, it was proposed that full-length KaiC from S. elongatus PCC 7942 (KaiC) is a member of the bacterial RecA/DnaB family (6). RecA is an ATP-dependent DNA recombinase, and DnaB is the replication fork helicase in bacteria. The kaiC gene appears to be an internally duplicated version of a RecA/DnaB-like gene; it has two parts that are very similar (7). In each half of the kaiC gene, there is a Walker A motif that binds ATP; when the Walker A motifs are mutated, nucleotide binding is abolished, and rhythmicity is severely disrupted or abolished (3, 8). At this time, the enzymatic function (if any) of KaiC is unknown, but its membership in the RecA/DnaB superfamily suggests a function relating to DNA (3).
We undertook a study using biophysical methods and electron microscopy (EM) to ascertain whether the sequence similarity between the structures of KaiC and RecA/DnaB was manifested in a structural relationship. We used KaiC from S. elongatus (optimal growth temperature 30Ò35?C), herein called "KaiC," and also from the mildly thermophilic cyanobacterium Synechococcus lividus (optimal growth temperature 50Ò55?C), herein called "KaiC-P2." We found that both of these KaiCs associate as homohexamers, dependent on ATP. Finally, both KaiCs bind forked DNA substrates despite the fact that neither of their sequences includes the DNA-binding motifs found in other members of the RecA/DnaB gene family.
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