Article activity feed

  1. Author Response:

    Reviewer #1 (Public Review):

    The manuscript by Gaubitz et al. reports structures of the yeast clamp loader (RFC)-sliding clamp (PCNA) complex in 6 different states during the clamp loading cycle. Although structures of yeast, human, E. coli and T4 clamp-loader-clamp complexes have been determined previously in various states, a major advance of the authors' work is to obtain structures of distinct intermediates in a single system. These structures provide a detailed description of the conformational changes in both RFC and PCNA during clamp loading, explaining ordered PCNA and primer/template DNA binding by RFC, the mechanism of clamp opening and closing, and the regulation of RFC's ATPase activity. In addition, the structures reveal differences in the mode of primer/template recognition between yeast and T4/E. coli clamp loaders. RFC melts the final base pair of the primer/template duplex using a separation pin in RFC-A, which is not seen in T4 or E coli clamp loaders. The authors confirm this interesting and unexpected observation biochemically. Although the authors speculate this mechanism could be used for distinguishing primer/template substrates from other DNA structures, the physiological significance of DNA melting and base flipping by RFC remains unclear. Overall, the findings reveal new nuances of the clamp loading cycle but the manuscript could be strengthened by solidifying the importance of base flipping for substrate recognition and RFC function.

    We thank the reviewer for their support. As mentioned above, we report new experiments examining the role of base-flipping residues on DNA binding and cell physiology (Figure 6 – Figure Supplements 2&3)

    Reviewer #2 (Public Review):

    In this study, Gausman et al. use cryo-electron microscopy to elucidate structures of complexes between the eukaryotic clamp loader (RFC) and its ligands, the DNA polymerase processivity clamp (PCNA) and DNA. Clamp loaders and clamps are required for DNA replication and repair in all domains of life. Understanding of the molecular mechanisms of clamp loading is not only important for DNA replication and repair, but also because clamp loaders are members of a larger group of motor proteins which are critical to many aspects of cellular metabolism. To date, our structural understanding of clamp loader mechanisms is based on comparison of structures for different clamp loader-ligand intermediate complexes from a variety of organisms including E. coli, yeast, bacteriophage, and humans. This paper presents the first structural data for multiple clamp loader-ligand intermediate complexes from a single organism, Saccharomyces cerevisiae, and sheds new light on protein-ligand interactions. Importantly, this work highlights structural features of the clamp loader that give rise to the order of ligand binding where the clamp loader binds and opens the clamp before binding DNA.

    To capture clamp loader ligand complexes, RFC was bound to the slowly hydrolyzable ATP analog, ATPγS, and intermediate complexes were further stabilized by protein crosslinking, predominantly intramolecular crosslinking of RFC subunits. Two types of RFC-PCNA complexes were observed, one in which the PCNA ring closed and a second where it is open. A family of closed complexes was observed in which three of the five RFC subunits contact the surface of the PCNA ring. Rigid body modeling suggests that this closed complex is dynamic such that the plane of the ring 'swings' relative to the clamp loader to potentially allow all five clamp loader subunits to engage the clamp to open the ring. In the open complex, the diameter of the complex expands and the opening in the PCNA ring is large enough to allow ds DNA to enter the ring and the chamber formed by the RFC subunits. A large hinge-like conformational change in the RFC-A subunit on going from closed to open complexes creates a channel for the ssDNA template to bind and exit the chamber. These remarkable structures show that the clamp loader is not in a suitable conformation to bind DNA prior to forming an open clamp complex which favors clamp binding before DNA binding.

    This manuscript provides remarkable insight into intermediate complexes that exist in the clamp loading reaction pathway. Having a family of structures for a single clamp loader and clamp provides a clearer picture of and highlights differences in clamp loading mechanisms from different organisms. Overall, this work well done, but perhaps some of the mechanistic conclusions drawn from static structures should be viewed with caution in the absence of rigorous dynamic or kinetic approaches.

    1. Crosslinking the proteins to stabilize intermediates could potentially bias the pool of conformations that are observed.

    We agree that crosslinking can bias the population of conformations observed. That is one of the reasons why we refrain from interpreting the number of particles in each class as being representative of the actual population of that intermediate.

    1. A statistical analysis of the differences in the ATPase activities of wild-type and mutant clamp loaders would be helpful to determine whether the mutations have an effect on the activity. Moreover, steady-state ATPase activity was measured in this experiment and these rates may not reveal differences in rates of intermediate steps in the clamp loading reactions. For the mutations to affect the ATPase activity, they would have to either change the rate of the rate-limiting step in the pathway or change the identity of the rate-limiting step. Thus, the decrease in ATPase activity for W638G mutant could be interesting if statistically significant.

    As mentioned above, we now report this analysis and interpretation in more detail.

    1. Given that Phe-582 and Trp-638 seem to be important for binding DNA at the 3' end, an analysis of the effects of mutations to these residues on DNA binding activity would be informative.

    As mentioned above, we report experiments examining these residues on DNA binding (Figure 6 – Figure Supplement 1) and new experiments measuring ATPase activity in the presence of various DNA substrates (Figure 6 – Figure Supplement 2).

    1. Kinetic data in the literature support a mechanism in which the clamp loader hydrolyzes ATP prior to clamp closing. In the absence of supporting kinetic data, it may be overinterpreting structural data to assert that the clamp loader need not hydrolyze ATP prior to closing the clamp.

    As mentioned above in detail, we agree and have toned down the interpretation.

    Was this evaluation helpful?
  2. Evaluation Summary:

    This work reports several cryoEM structures of clamp loader-sliding clamp complexes, which are required for DNA replication and repair in all domains of life, and is of interest to researchers studying DNA metabolism and motor proteins. The findings provide new insight into the mechanism of clamp loading and the mechanisms by which ligands affect the conformational dynamics of motor proteins to facilitate their reactions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

    Was this evaluation helpful?
  3. Reviewer #1 (Public Review):

    The manuscript by Gaubitz et al. reports structures of the yeast clamp loader (RFC)-sliding clamp (PCNA) complex in 6 different states during the clamp loading cycle. Although structures of yeast, human, E. coli and T4 clamp-loader-clamp complexes have been determined previously in various states, a major advance of the authors' work is to obtain structures of distinct intermediates in a single system. These structures provide a detailed description of the conformational changes in both RFC and PCNA during clamp loading, explaining ordered PCNA and primer/template DNA binding by RFC, the mechanism of clamp opening and closing, and the regulation of RFC's ATPase activity. In addition, the structures reveal differences in the mode of primer/template recognition between yeast and T4/E. coli clamp loaders. RFC melts the final base pair of the primer/template duplex using a separation pin in RFC-A, which is not seen in T4 or E coli clamp loaders. The authors confirm this interesting and unexpected observation biochemically. Although the authors speculate this mechanism could be used for distinguishing primer/template substrates from other DNA structures, the physiological significance of DNA melting and base flipping by RFC remains unclear. Overall, the findings reveal new nuances of the clamp loading cycle but the manuscript could be strengthened by solidifying the importance of base flipping for substrate recognition and RFC function.

    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    In this study, Gausman et al. use cryo-electron microscopy to elucidate structures of complexes between the eukaryotic clamp loader (RFC) and its ligands, the DNA polymerase processivity clamp (PCNA) and DNA. Clamp loaders and clamps are required for DNA replication and repair in all domains of life. Understanding of the molecular mechanisms of clamp loading is not only important for DNA replication and repair, but also because clamp loaders are members of a larger group of motor proteins which are critical to many aspects of cellular metabolism. To date, our structural understanding of clamp loader mechanisms is based on comparison of structures for different clamp loader-ligand intermediate complexes from a variety of organisms including E. coli, yeast, bacteriophage, and humans. This paper presents the first structural data for multiple clamp loader-ligand intermediate complexes from a single organism, Saccharomyces cerevisiae, and sheds new light on protein-ligand interactions. Importantly, this work highlights structural features of the clamp loader that give rise to the order of ligand binding where the clamp loader binds and opens the clamp before binding DNA.

    To capture clamp loader ligand complexes, RFC was bound to the slowly hydrolyzable ATP analog, ATPγS, and intermediate complexes were further stabilized by protein crosslinking, predominantly intramolecular crosslinking of RFC subunits. Two types of RFC-PCNA complexes were observed, one in which the PCNA ring closed and a second where it is open. A family of closed complexes was observed in which three of the five RFC subunits contact the surface of the PCNA ring. Rigid body modeling suggests that this closed complex is dynamic such that the plane of the ring 'swings' relative to the clamp loader to potentially allow all five clamp loader subunits to engage the clamp to open the ring. In the open complex, the diameter of the complex expands and the opening in the PCNA ring is large enough to allow ds DNA to enter the ring and the chamber formed by the RFC subunits. A large hinge-like conformational change in the RFC-A subunit on going from closed to open complexes creates a channel for the ssDNA template to bind and exit the chamber. These remarkable structures show that the clamp loader is not in a suitable conformation to bind DNA prior to forming an open clamp complex which favors clamp binding before DNA binding.

    This manuscript provides remarkable insight into intermediate complexes that exist in the clamp loading reaction pathway. Having a family of structures for a single clamp loader and clamp provides a clearer picture of and highlights differences in clamp loading mechanisms from different organisms. Overall, this work well done, but perhaps some of the mechanistic conclusions drawn from static structures should be viewed with caution in the absence of rigorous dynamic or kinetic approaches.

    1. Crosslinking the proteins to stabilize intermediates could potentially bias the pool of conformations that are observed.

    2. A statistical analysis of the differences in the ATPase activities of wild-type and mutant clamp loaders would be helpful to determine whether the mutations have an effect on the activity. Moreover, steady-state ATPase activity was measured in this experiment and these rates may not reveal differences in rates of intermediate steps in the clamp loading reactions. For the mutations to affect the ATPase activity, they would have to either change the rate of the rate-limiting step in the pathway or change the identity of the rate-limiting step. Thus, the decrease in ATPase activity for W638G mutant could be interesting if statistically significant.

    3. Given that Phe-582 and Trp-638 seem to be important for binding DNA at the 3' end, an analysis of the effects of mutations to these residues on DNA binding activity would be informative.

    4. Kinetic data in the literature support a mechanism in which the clamp loader hydrolyzes ATP prior to clamp closing. In the absence of supporting kinetic data, it may be overinterpreting structural data to assert that the clamp loader need not hydrolyze ATP prior to closing the clamp.

    Was this evaluation helpful?