|Protein Structure Suggests Role as Molecular Adapter|
To split and copy DNA during replication, all cellular organisms use a multicomponent molecular machine known as the replisome. An essential step in replisome assembly is the loading of ring-shaped helicases (motor proteins) onto the separated strands of DNA. Dedicated ATP-fueled proteins regulate the loading; however, the mechanism by which these proteins recruit and deposit helicases has remained unclear. To better understand this process, researchers at the University of California, Berkeley, recently determined the structure of the ATPase region of DnaC, a bacterial helicase loader. The structure revealed that DnaC is a close cousin of DnaA, the protein thought to be responsible for unwinding DNA. Unexpectedly, the team further found that DnaC forms a right-handed helix similar to the state adopted by ATP-bound DnaA. These findings, together with biochemical studies, implicate DnaC as a molecular adapter that uses ATP-activated DnaA as a docking site for ensuring that DnaB (the ring-shaped helicase) is correctly deposited at the onset of replication.
Recent structural studies revealed that ATP promotes the oligomerization of DnaA into helical filaments, an organization that may play a direct role in separating DNA strands. By contrast, little was known about how the ATPase domain of DnaC works with the ATP-activated DnaA. Therefore, the team investigated the molecular structure of the ATPase domain of DnaC from the bacterium Aquifex aeolicus at ALS crystallography Beamline 8.3.1. The structure, solved to 2.7-Å resolution, established that the loader (DnaC) and initiator (DnaA) are closely related structurally and allowed the researchers to visualize active-site residues responsible for ATP binding in DnaC that were previously unclear from sequence alignments alone. Significantly, the structural congruence between DnaC and DnaA extends to the ability of the helicase loader to assemble into a right-handed helical oligomer, a structural state highly similar to that previously observed for ATP-bound DnaA.
Using the structure as a guide, the researchers went on to show that contacts between DnaC subunits in the protein crystal were critical for helicase-loader function in vitro and in vivo. They also uncovered an unexpected interaction between DnaC and DnaA that is manifested through the ATPase domains of the two proteins and dependent on ATP. Together, these observations led to a new model that helps explain how two copies of the replicative helicase, DnaB, are properly positioned and oriented on the complementary single strands of DNA formed by the replisome.
The process is as follows: Following separation of the DNA strands, the DnaA helix is believed to associate predominantly with the "upper" strand of the newly opened region. DnaA then recruits and helps load a single DnaB–DnaC complex onto the lower strand, an event facilitated by the N-terminal domain of the initiator (DnaA), which is known to bind the N-terminal side of the helicase (DnaB). DnaB is loaded onto the upper strand through the DnaC—DnaA interaction. Interestingly, the N-terminal region of the loader (DnaC) is known to interact with the C-terminal face of DnaB. This arrangement suggests that the observed DnaA–DnaC interaction reverses the orientation of one of the DnaB helicases, a necessary event because the DnaB helicase — a motor protein — must travel to the right in the upper strand and to the left on the lower strand.
Collectively, these findings provide a new role for DnaC beyond merely opening a DnaB ring to permit loading onto single-stranded DNA. In particular, the data support an unanticipated role for DnaC as an adapter that can specifically recognize an ATP-activated DnaA initiator, most likely through direct heterologous ATPase domain interactions. Given the ubiquity of ATPases in controlling initiation in eukaryotic as well as bacterial cells, this mechanism for generating a symmetric, bidirectional replication fork from an asymmetric initiator complex may have important functional parallels throughout all domains of life.
Research conducted by M.L. Mott, M.M. Coons, and J.M. Berger (University of California, Berkeley) and J.P. Erzberger (Swiss Federal Institute of Technology, Zurich).
Research funding: National Science Foundation and National Institutes of Health. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences.
Publication about this research: M.L. Mott, J.P. Erzberger, M.M. Coons, and J.M. Berger, "Structural synergy and molecular crosstalk between bacterial helicase loaders and replication initiators," Cell 135, 623 (2008).