Stepwise assembly of the human replicative polymerase holoenzyme

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In this paper Benkovic and colleagues analyze the interaction between the DNA polymerase clamp, PCNA, the clamp loader (RFC), and primer-template DNA. The manuscript describes a series of carefully designed and closely argued FRET experiments that seek to dissect the detailed conformational steps in the loading and unloading of human PCNA by human RFC. The advance reported in this paper concerns the role of the RFC complex in unloading PCNA from DNA after it has been loaded.

As the authors point out, it is well understood that PCNA does not readily load on to DNA on its own, and that once loaded it is very stable on DNA because it forms a closed ring. It is also appreciated, from previous work, that clamp loaders can unload clamps from DNA, in the reverse of the reaction that puts them on to DNA, which helps recycling clamps from Okazaki fragments.

Hedglin et al. now present a kinetic analysis of human RFC focused on both the clamp loading and unloading phases of the reaction. The authors delineate distinct ATP binding and ATP hydrolysis steps in the loading reaction, and show that PCNA transiently released from RFC following ATP hydrolysis can be recaptured by RFC in the first step of an unloading reaction. At one level, this is not surprising, and reflects what would be predicted by micro-reversibility of an overall equilibrium process. Nevertheless, the major conclusion, which is that RFC does not dissociate after loading PCNA and can unload the clamp from DNA, runs counter to the prevailing view and therefore increases interest in the paper. The authors suggest that RFC efficiently loads and unloads clamps at primed DNA within a single binding encounter with the primer-template junction. This has major biological implications as it would lower the efficiency of clamp loading during DNA replication, when the required loading rate is ∼1 clamp/second. The problem would be acute if there were any lag between clamp loading and polymerase arrival. Thus, the conclusions of this study need to be carefully validated because they have the potential to change our understanding of a fundamental aspect of this important step in DNA replication.

There are two major concerns about the generality of the conclusions drawn from the experiments reported in this paper, both of which have to do with the nature of the DNA construct used. The experiments are well performed, and the reasoning behind the experiments is well explained, but the experiments all rely on a particular DNA construct, shown in Figure 1. This construct is designed to block PCNA sliding off the DNA in two ways. First, streptavidin is bound to a biotin tag on one end. Second, a single-stranded overhang is present at the other end, which also provides a block for PCNA sliding. The two major concerns are as follows:

1) Various clamp loaders, such as E. coli γ complex, as well as human and yeast RFC are known to bind single stranded DNA with high affinity. Does the 5' ssDNA flap shift the equilibrium towards clamp unloading by providing a binding site for RFC and enabling it to remain close to loaded PCNA? If there were no such extra binding sites for RFC (no ssDNA in the substrate or the presence of RPA), would there be any significant clamp unloading in a single cycle?

2) From a large body of previously published work we know that polymerases and clamp loaders compete for the same binding sites on the clamp, and so it is not surprising that the arrival of the polymerase (Polδ is used in the experiments reported here) prevents RFC from unloading PCNA. But, in the context of the experiments described here, there is difficulty in understanding how Polδ accesses the same face of the PCNA clamp as does RFC, if a transient association between RFC and PCNA is being maintained. There is a concern that the distance between the RFC loading site and the biotin-avidin block used in this study overly restricts the degree to which the released PCNA can truly dissociate from RFC. Is RFC moving away from the double-helical region and being held by the single-stranded DNA, as discussed in point 1, above? If so, would such an interaction be physiologically relevant?

In light of these two concerns, the paper would be greatly strengthened if the authors explored (A) the effect of removing the single-stranded overhang or the addition of single-stranded DNA binding protein, e.g., RPA and (B) the effect of longer spacings between the P/T junction and the biotin-avidin block to properly define the extent of dissociation that is needed for Polδ to intervene, and the effect of PCNA diffusion away from the loading site on the kinetics of re-binding to RFC in the unloading process. In the absence of experiments on variants of the DNA construct used, there would be lingering questions about whether the results are an artifact of RFC and PCNA being trapped by a DNA construct that differs in important ways from the structure of a replication fork. We recognize that certain aspects of the DNA construct, such as the flap, may not easily be changed, but if these concerns cannot be overcome then the findings could be open to alternative interpretations.