Clamp loaders perform the essential task of loading circular clamps onto DNA where they are utilized in DNA replication and repair, as well as several other cellular processes. This task requires the clamp loader to direct transient conformational changes and interactions among the reaction components towards formation of a topologically linked clamp•DNA product. For example, early in the reaction the clamp loader, clamp, and DNA must develop high affinity for each other, adopting conformations that enable positioning of DNA within the clamp, and later their interactions must change to allow the release of clamp•DNA. ATP binding and hydrolysis catalyzed by the clamp loader promote and coordinate events in this intricate reaction mechanism. Past investigation of the eukaryotic S. cerevisiae
RFC clamp loader has revealed that it binds both PCNA clamp and primed-template DNA in the presence of non-hydrolyzable ATPγS 23,24,26
. ATPγS-bound RFC is also capable of opening PCNA, possibly enough to allow entry of duplex DNA into the center of the ring 17,24
. In this study, we aimed to elucidate the mechanism of RFC-catalyzed PCNA loading by measuring the kinetics of several transactions, including ATP binding/hydrolysis, DNA binding, PCNA opening/closing and product release that occur during the reaction.
The ATPase reaction promotes two distinct phases, one that involves assembly of an RFC•ATP•PCNA•ptDNA complex, and another that involves disassembly of the complex. Our kinetic data show directly that complex assembly is coupled to ATP binding and disassembly is coupled to ATP hydrolysis (). RFC has five nucleotide binding sites, three of which are occupied by ATPγS in the absence of PCNA and ptDNA substrates, while the remaining two are occupied when RFC binds PCNA or ptDNA or both (; schematic depiction in ). A previous report suggested very different stoichiometry, with only four ATPγS molecules binding per RFC—two to free RFC, one more to RFC•ATP•PCNA, and fourth one to RFC•ATP•PCNA•ptDNA complex 23
. However, the clamp loader examined in that study contained an N-terminal deletion of 269 amino acids from the RFC-A (Rfc1) subunit. Our analysis of a similar N-terminal truncated clamp loader, missing 282 amino acids from RFC-A, also indicates that this mutant (ΔN-RFC) binds fewer ATPγS molecules than full length RFC (data not shown) and, significantly, the pre-steady state burst ATPase activity catalyzed by ΔN-RFC is also lower, indicating that the activity of this mutant deviates from that of the wild type clamp loader (Supplementary Material, Figure S2
). The crystal structure of RFC•ATPγS•PCNA complex shows nucleotides bound at all five sites 8
Pre-steady state ATPase data reveal important aspects of the assembly phase of the reaction. First, comparison of the ATPase kinetics in the absence and presence of primed-template DNA shows an absolute requirement for ptDNA binding in order for RFC to catalyze rapid ATP hydrolysis (, , ). Thus, ATP-bound RFC remains in a practically ‘ATPase-inactive’ conformation until ptDNA swiftly switches it to a conformation that is competent for hydrolysis. This DNA-dependent switch occurs whether or not RFC is in complex with PCNA; however, the ATPase burst is substantially larger in the presence of PCNA than in its absence ( versus ). Specifically, global analysis of the data suggests that RFC with PCNA hydrolyzes more ATP and at a faster rate than without PCNA (, NATP = 3 versus 2 and kR(A)DNA_ATPase = 54 s−1 versus 11 s−1). These differences in ATPase stoichiometry and kinetics could reflect distinct properties of the RFC•ATP•PCNA and RFC•ATP complexes. They could also arise from a larger fraction of RFC•ATP•PCNA achieving ‘ATPase-active’ conformation compared with RFC•ATP following ptDNA binding, or from other steps in the reactions not measured explicitly yet (e.g., differential changes in RFC conformation in these two complexes). Alternately, these complexes may hydrolyze additional ATP molecules at relatively slow rates that are not detected in the ATPase burst phase. In any case, the data reveal a step that blocks RFC ATPase activity in the first phase of the reaction, presumably to allow assembly of RFC, ATP and PCNA into a complex with minimal futile ATP hydrolysis, until ptDNA binding triggers the second, disassembly phase of the reaction.
We also discovered a slow RFC activation step that occurs after ATP binding (). This step occurs at a faster rate of 4.6 s−1
in the presence of PCNA compared with 1.5 s−1
in the absence of PCNA (, ; kR_Activation
). During the activation period RFC binds and opens PCNA, generating an RFC•ATP•PCNA(open)
intermediate complex that can allow entry of ptDNA into the clamp (). ATP binding-induced RFC activation appears absolutely necessary for its interaction with ptDNA leading to rapid ATP hydrolysis ( and ). Crystal structures of S. cerevisiae
RFC and other clamp loaders suggest that ATP binding drives the clamp loader subunits into a spiral arrangement such that their DNA binding sites can track the duplex portion of ptDNA when it enters the complex 4,8,15
. PCNA docks under RFC and is predicted to open in a spiral conformation complementary to that adopted by the clamp loader subunits, which could explain how it stimulates activation of RFC 14,17,30
. That RFC activation occurs at a faster rate when the clamp loader is in complex with PCNA increases the likelihood that during DNA replication in vivo
, with high concentrations of PCNA and limited primed-template sites, ptDNA will bind a pre-assembled RFC•ATP•PCNA(open)
complex rather than RFC•ATP complex, resulting in a productive PCNA loading reaction cycle.
Once the ptDNA is bound, RFC conformation changes to enable ATP hydrolysis and initiate the second, disassembly phase of the reaction. In our minimal RFC mechanism, we sorted dissociation of the four reaction products, ADP, Pi, PCNA, and ptDNA into two distinct steps, assuming that either ADP and Pi or ptDNA and PCNA leave the complex first (). Global analysis of the ATPase data indicates that dissociation of PCNA•ptDNA from RFC occurs at a rate of 1.7 s−1, which is slow compared to dissociation of ptDNA alone at 25 s−1 (, ; kR-DNA_Off). Dissociation rates for each of these products have to be measured and incorporated into the model before we can determine explicitly which step(s) controls the catalytic turnover rate of RFC. Based on the parameters obtained from ATPase data fitting, we can estimate a steady state velocity of 3.5 μM s−1 for the reaction containing PCNA and ptDNA (kcat = 1.2 s−1 for 3 sites or 0.9 s−1 for 4 ATPase sites per RFC). There are several slow steps following rapid ATP hydrolysis (54 s−1), including PCNA closing (~ 5 s−1, ), Pi release (6.4 s−1), PCNA•ptDNA release (1.7 s−1), and the next round of RFC activation (4.6 s−1), which likely limit the steady state clamp loading rate ().
Our results indicate that RFC can load and release PCNA onto a primed-template site on DNA at a rate of about 1 s−1, which is compatible with the estimated rate of Okazaki fragment synthesis in vivo (0.5 – 1 s−1). Thus, PCNA clamps can be loaded in time for DNA polymerase to cycle efficiently from completing an Okazaki fragment to beginning processive synthesis of a new one. It is also noteworthy that in the absence of PCNA, RFC activation limits the start of the reaction, and ptDNA bound to RFC under these conditions undergoes rapid dissociation at the end of the reaction. These kinetics reflect a preferred order of events in the clamp loading reaction, with RFC slow to bind ptDNA without PCNA; however, if it does happen to bind ptDNA ahead of PCNA, ATP hydrolysis and ptDNA release quickly reset the clamp loader to the beginning of the cycle.
This study reveals some common features between the reaction mechanisms of S. cerevisiae
RFC and E. coli
γ complex (γ3
δδ′χψ) clamp loaders. The ATPase kinetics of γ complex also indicate a slow change in conformation following ATP binding and prior to ATP hydrolysis, which activates the clamp loader for interaction with DNA 5,20
. The presence of β initially suppresses ATP hydrolysis by γ complex and then enhances ATP hydrolysis following addition of ptDNA, consistent with the proposition that early rate limiting steps in the ATPase reaction favor a productive clamp loading reaction. Binding of ptDNA to γ complex•ATP•β triggers rapid ATP hydrolysis at the 3 γ subunits and release of β•ptDNA to complete the reaction. Intriguingly, analysis of the RFC ATPase data based on the minimal mechanism in also indicates that 3 ATP molecules are hydrolyzed rapidly by RFC in one turnover (; NATP
= 3.1). One can therefore consider the possibility that hydrolysis of 3 ATP may be sufficient for ATPase-coupled loading of clamps onto DNA. The E. coli
γ complex contains only 3 ATPase-active γ subunits, with the homologous δ′ and δ subunits having lost their ability to bind and hydrolyze ATP 4
. In the case of RFC, Walker ATP binding and hydrolysis motifs are disrupted in RFC-E subunit (Rfc5), whose position in RFC is analogous to that of δ′ in γcomplex, rendering it unable to hydrolyze ATP (although it is still capable of binding ATP; ) 8
. The RFC-A subunit (Rfc1), whose position in RFC is analogous to that of δ in γcomplex, has an apparently functional ATPase site, but according to an in vivo
study its ATPase activity does not appear to be essential for PCNA loading 31
; the same study found that the ATPase activities of RFC-B, RFC-C and RFC-D subunits (Rfc4, Rfc3, Rfc2) are essential for PCNA loading.
It remains possible that a sub-stoichiometric fraction of RFC is active (or activated) for ATP hydrolysis in our experiments, despite being fully active for ATPγS binding. Alternately, hydrolysis of a fourth ATP by RFC may occur at a different rate, possibly associated with another step in the reaction. The latter scenario is analogous to that proposed for archaeal A. fulgidus
RFC, which also has 4 ATPase-active subunits, and in this case the activity of 3 subunits appears necessary up to PCNA•DNA release and that of a fourth subunit affects the turnover rate 16
. The bacteriophage T4 gp44/62 clamp loader also has 4 ATPase-active subunits and the reaction kinetics indicate similarities with γ complex and RFC mechanisms, such as the requirement of ATP hydrolysis for gp45 clamp and ptDNA release from gp44/62, but also some key differences 13,22
. For example, gp44/62 is reported to rapidly hydrolyze 2 ATP molecules in the presence of gp45 alone, 1 ATP in the presence of ptDNA alone, and all 4 ATP molecules in the presence of both gp45 and ptDNA 21,22
. It has been proposed that the T4 clamp loader utilizes alternate pathways for clamp assembly on DNA (perhaps the open gp45 clamp affords more mechanistic flexibility than the closed β and PCNA clamps), and that the stoichiometry and pattern of ATP hydrolysis varies with the pathway 12,21
. In the case of RFC, there is no specific evidence for multiple clamp assembly pathways, but additional kinetic analysis, for example of RFC complexes containing individual ATPase-inactive subunits, can further resolve the question of how ATP is utilized in the clamp loading reaction.
In summary, our findings have provided direct evidence for an ordered series of ATP binding and hydrolysis-coupled events that drive clamp assembly on ptDNA by a eukaryotic clamp loader. Important questions remain regarding the mechanism of RFC and related eukaryotic clamp loaders, especially pertaining to the transactions of PCNA and ptDNA in the reaction and the nature of the conformational changes in RFC. Intriguing questions arise also from studies of more distant clamp loaders in the superfamily, such as the possible existence of multiple clamp assembly pathways. The discovery and quantitative description of key steps in the S. cerevisiae RFC-catalyzed reaction provide useful leads for focused inquiry into the workings of this essential DNA metabolic protein in multiple organisms.