Clamp loaders load ring-shaped sliding clamps onto DNA. Once loaded onto DNA, sliding clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load clamps onto DNA, an open clamp loader-clamp complex must form. An unresolved question is whether clamp loaders capture clamps that have transiently opened or whether clamp loaders bind closed clamps and actively open clamps. A simple fluorescence-based clamp opening assay was developed to address this question and to determine how ATP binding contributes to clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds β first and then opens the clamp. Mutation of conserved “arginine fingers” in the γ complex that interact with bound ATP decreased clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the clamp loader to pry open the clamp.

The human alkyladenine DNA glycosylase has a broad substrate specificity, excising a structurally diverse group of damaged purines from DNA. To more clearly define the structural and mechanistic bases for substrate specificity of human alkyladenine DNA glycosylase, kinetics of excision and DNA binding activities were measured for several different damaged and undamaged purines within identical DNA sequence contexts. We found that1,N ⁶-ethenoadenine (εA) and hypoxanthine (Hx) were excised relatively efficiently, whereas 7,8-dihydro-8-oxoguanine, O ⁶-methylguanine, adenine, and guanine were not. Single-turnover kinetics of excision of Hx and εA paired with T showed that excision of Hx was about four times faster than εA, whereas binding assays showed that the binding affinity was about five times greater for εA than for Hx. The opposing pyrimidine base had a significant effect on the kinetics of excision and DNA binding affinity of Hx but a small effect on those for εA. Surprisingly, replacing a T with a U opposite Hx dramatically reduced the excision rate by a factor of 15 and increased the affinity by a factor of 7–8. The binding affinity of human alkyladenine DNA glycosylase to a DNA product containing an abasic site was similar to that for an Hx lesion.

Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, χ and ψ, which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, γ₃δδ′, γ₃δδ′ψ, and γ₃δδ′ψχ. The ψ subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the χ subunit does not contribute directly to clamp loading in our assays lacking single-stranded DNA-binding protein. The ψ subunit also increases the affinity of the clamp loader for the clamp in assays in which ATPγS is substituted for ATP. Interestingly, the affinity of the γ₃δδ′ complex for β is no greater in the presence than in the absence of ATPγS. A role for ψ in stabilizing or promoting ATP- and ATPγS-induced conformational changes may explain why large conformational differences were not seen in γ₃δδ′ structures with and without bound ATPγS. The β clamp partially compensates for the activity of ψ when this subunit is not present and possibly serves as a scaffold on which the clamp loader adopts the appropriate conformation for DNA binding and clamp loading. Results from our work and others suggest that the ψ subunit may introduce a temporal order to the clamp loading reaction in which clamp binding precedes DNA binding.

Clamp loaders from all domains of life load clamps onto DNA. The clamp tethers DNA polymerases to DNA to increase the processivity of synthesis as well as the efficiency of replication. Here, we investigated proliferating cell nuclear antigen (PCNA) binding and opening by the Saccharomyces cerevisiae clamp loader, replication factor C (RFC), and the DNA damage checkpoint clamp loader, Rad24-RFC, using two separate fluorescence intensity-based assays. Analysis of PCNA opening by RFC revealed a two-step reaction in which RFC binds PCNA before opening PCNA rather than capturing clamps that have transiently and spontaneously opened in solution. The affinity of RFC for PCNA is about an order of magnitude lower in the absence of ATP than in its presence. The affinity of Rad24-RFC for PCNA in the presence of ATP is about an order magnitude weaker than that of RFC for PCNA, similar to the RFC-PCNA interaction in the absence of ATP. Importantly, fewer open clamp loader-clamp complexes are formed when PCNA is bound by Rad24-RFC than when bound by RFC.