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Brian received his PhD from the Department of Biochemistry & Biophysics at UCSF in 2007, where he studied the folding mechanism of a class of ultrastable proteases with David Agard. He was a NRSA fellow with John Kuriyan at UC Berkeley where he studied the structural mechanisms of macromolecular machines involved in DNA replication. He joined the Department of Biochemistry & Molecular Pharmacology in 2012.
Structure & Mechanism of DNA replication machines
The major focus of my lab is determining the structural mechanisms of the macromolecular machines that carry out DNA replication.
Macromolecular machines are assemblies of proteins and/or nucleic acids that play key roles in DNA replication, transcription, translation, virus maturation, and many other cellular functions. Accordingly, they are important therapeutic targets for the development of novel anticancer and antiviral agents. However, understanding how the individual components operate together is often elusive.
The Kelch laboratory takes an interdisciplinary approach to study whole macromolecular machines and determine their mechanism.
Clamp Loaders & DNA replication
High-speed replication of chromosomal DNA requires the DNA polymerase to be attached to a ring-shaped sliding clamp that encircles DNA and prevents the polymerase from falling off DNA. Sliding clamps are crucial for DNA replication, DNA repair, cell cycle control and modification of chromatin structure. Defects in several clamp-associated factors are associated with cancer and other disorders.
The clamp loader, a member of AAA+ family of ATPases, opens sliding clamps and places them on DNA for polymerase to bind. Thus, the clamp loader is critical for the tight coupling of leading and lagging strand synthesis. In order to gain an understanding of the mechanism of the clamp loading reaction at the atomic level, we determined the structure of the clamp loader in complex with its two macromolecular substrates: the sliding clamp and primer-template DNA (Figure 1).
|Figure 1: Structure of the complex of clamp loader, sliding clamp, and primer-template DNA (Kelch et al Science 2011).|
Based on our recent structural analysis of intermediates in clamp loader reaction cycle (Kelch et al Science 2011; Kelch et al BMC Bio 2012), we have proposed a detailed model of the clamp loading mechanism, as described in Figure 2. The clamp loader traps a spiral conformation of the open clamp so that both the loader and the clamp match the helical symmetry of DNA. The symmetric spiral of the clamp loader is dependent on the binding of ATP at the interfaces between AAA+ modules of the clamp loader. We propose that ATP hydrolysis breaks the symmetric spiral of the AAA+ modules, leading to the clamp loader dissolving its contacts with both the clamp and the DNA.
Figure 2: Proposed mechanism of the clamp loading cycle.
Action of enzymes on sliding clamps
Once the clamp is loaded onto DNA, it acts as a sliding platform for the action of scores of enzymes and other proteins to scan and act on DNA. The clamp acts as a central hub to organize the activities of enzymes in the various cellular pathways of DNA replication, DNA repair, cell cycle control, chromatin structure and apoptosis.
However, how these various activities are coordinated spatially and temporally is unknown. We want to know the cellular, molecular and structural determinants for this coordination.
We are developing model systems to understand the dynamics of enzymes on the clamp and to understand how their activity is regulated while on the sliding clamp.
Structural and mechanistic studies of alternative clamp loaders
Eukaryotic organisms have an array of alternative clamp loaders that are used in pathways distinct from DNA replication, such as DNA damage and checkpoint control, sister chromatid cohesion, and recombination (Figure 3). These clamp loaders have been suggested to be important targets for novel cancer therapeutics. Despite differing from the replicative clamp loader by just one subunit in the pentameric assembly, the alternative clamp loaders exhibit significant functional and mechanistic differences. For example the Rad24 clamp loader binds a different sliding clamp, the ’9-1-1 complex’ (Majka & Burgers, PNAS 2003), and functions at the 5’ DNA junction, the opposite polarity to that of the replicative clamp loader (Ellison & Stillman, PLOS Bio 2003).
Our goal is to determine the structural and biochemical basis for the mechanistic and functional differences of the alternative clamp loaders from their replicative clamp loader cousins. We will also understand how alternative clamp loaders are regulated and integrated into the pathways of recombination, sister chromatid cohesion and DNA repair. These studies will reveal how AAA+ machines can be regulated, as well as identifying the mechanistic underpinnings of these key components of important cellular pathways.
Figure 3: The function of the alternative clamp loaders