Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies

Kumar, Ravindra; Nashine, Vishal; Mishra, Padmaja P.; Benkovic, Stephen J.; Lee, Tae Hee

doi:10.1073/pnas.1014139107

Cited by 25 publications

(35 citation statements)

References 42 publications

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“…1 s −1 (Supplementary Table S1). Biphasic kinetic behavior has been reported for the eukaryotic clamp loading and unloading reactions, and the data were interpreted as reflecting two distinct populations of clamp/clamp loader/DNA ternary complexes, one of which can go on to form active initiation complexes 36 . The double exponential behavior observed here with the E. coli holoenzyme could reflect a similar competition between reaction pathways that form incomplete or inactive sub-assemblies of holoenzyme components on the DNA and pathways that form initiation complexes active for DNA synthesis.…”

Section: Resultsmentioning

confidence: 95%

“…The eukaryotic clamp loading process is rapid (rate constant as fast as ca. 10 s −1 36 ), but no binding interactions have been identified between the eukaryotic clamp loader and polymerases, and the mechanism by which the polymerases associate with the loaded clamp is not well understood. While it is clearly important that clamp loading be fast, what ultimately matters for replication fork progression is the rate for completing initiation complex formation.…”

Section: Discussionmentioning

confidence: 99%

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Polymerase Chaperoning and Multiple ATPase Sites Enable the E. coli DNA Polymerase III Holoenzyme to Rapidly Form Initiation Complexes

Downey

Crooke

McHenry

2011

Journal of Molecular Biology

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Cellular replicases include three subassemblies: a DNA polymerase, a sliding clamp processivity factor, and a clamp loader complex. The E. coli clamp loader is the DnaX complex (DnaX3δδ'χψ), where DnaX occurs either as τ or as the shorter γ that arises by translational frameshifting. Complexes comprised of either form of DnaX are fully active clamp loaders, but τ confers important replicase functions including chaperoning the polymerase to the newly loaded clamp to form an initiation complex for processive replication. The kinetics of initiation complex formation were explored for DnaX complexes reconstituted with varying τ and γ stoichiometries, revealing that τ-mediated polymerase chaperoning accelerates initiation complex formation 100-fold. Analyzing DnaX complexes containing one or more K51E variant DnaX subunits demonstrated that only one active ATP-binding site is required to form initiation complexes, but the two additional sites increase the rate ca.1000-fold. For τ-containing complexes, the ATP analogue ATPγS was found to support initiation complex formation at 1/1000th the rate with ATP. In contrast to previous models that ATPγS drives hydrolysis-independent initiation complex formation by τ-containing complexes, the rate and stoichiometry of ATPγS hydrolysis coincide with those for initiation complex formation. These results show that although one ATPase site is sufficient for initiation complex formation, the combination of polymerase chaperoning and the binding and hydrolysis of three ATPs dramatically accelerates initiation complex formation to a rate constant (25–50 s−1) compatible with double-stranded DNA replication.

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Section: Resultsmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Polymerase Chaperoning and Multiple ATPase Sites Enable the E. coli DNA Polymerase III Holoenzyme to Rapidly Form Initiation Complexes

Downey

Crooke

McHenry

2011

Journal of Molecular Biology

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“…The clamp loading reaction is quite complex in that at least a dozen steps are required to describe a simple linear reaction pathway [13,15,21,23,43–45]. These steps include binding 4 or 5 molecules of ATP, clamp binding, clamp opening, DNA binding, ATP hydrolysis, clamp closing, clamp release, DNA release, ADP/P i dissociation, and at least two conformational changes in RFC induced by ATP binding and hydrolysis.…”

Section: Discussionmentioning

confidence: 99%

Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C

Marzahn

Hayner

Meyer

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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DNA polymerases require a sliding clamp to achieve processive DNA synthesis. The toroidal clamps are loaded onto DNA by clamp loaders, members of the AAA+ family of ATPases. These enzymes utilize the energy of ATP binding and hydrolysis to perform a variety of cellular functions. In this study, a clamp loader-clamp binding assay was developed to measure the rates of ATP-dependent clamp binding and ATP-hydrolysis-dependent clamp release for the S. cerevisiae clamp loader (RFC) and clamp (PCNA). Pre-steady-state kinetics of PCNA binding showed that although ATP binding to RFC increases affinity for PCNA, ATP binding rates and ATP-dependent conformational changes in RFC are fast relative to PCNA binding rates. Interestingly, RFC binds PCNA faster than the Escherichia coli γ complex clamp loader binds the β-clamp. In the process of loading clamps on DNA, RFC maintains contact with PCNA while PCNA closes, as the observed rate of PCNA closing is faster than the rate of PCNA release, precluding the possibility of an open clamp dissociating from DNA. Rates of clamp closing and release are not dependent on the rate of the DNA binding step and are also slower than reported rates of ATP hydrolysis, showing that these rates reflect unique intramolecular reaction steps in the clamp loading pathway.

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“…The kinetics of gp45 loading onto DNA displayed a singlephase exponential association, and differs from the replication factor C-mediated loading of the proliferating cell nuclear antigen clamp that displayed biphasic exponential kinetics (29). The observed association rate of DNA and gp45-gp44/62-ATP was first order, indicating a rate-determining repositioning of gp45 after rapid binding, which is further supported by our single-molecule results revealing multistep rearrangement of gp45 on binding DNA before it can be stably loaded.…”

Section: Discussionmentioning

confidence: 99%

How a holoenzyme for DNA replication is formed

Perumal

Ren

Lee

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Loading of the phage T4 sliding clamp gp45 by the gp44/62 clamp loader onto DNA to form the holoenzyme and their disassembly pathways were investigated using FRET-based single-molecule and ensemble kinetic studies. gp44/62-mediated assembly of gp45 onto the DNA involves a rate-limiting conformational rearrangement of the gp45−gp44/62−DNA complex. Single-molecule measurements revealed the intermediates in gp45 loading and their interconversion, suggesting that the assembly is not concerted but is broken down into many small kinetic steps. Two populations of the gp45−gp44/62−DNA complex are formed on the end-blocked DNA that are poised to form the holoenzyme with the polymerase. In the absence of a polymerase, the two clamp populations dissociated from the DNA along with gp44/62 with distinct rates. In the presence of polymerase, holoenzyme assembly involved the recruitment of the polymerase to the gp45−gp44/62−DNA complex mediated by the chaperoning activity of gp44/62. This transient multiprotein complex then decomposed through an ATP hydrolysis−dependent exit of gp44/62 leaving the holoenzyme on DNA. The rate of dissociation of the holoenzyme from the DNA is sensitive to whether the DNA ends are blocked, underscoring its mobility on the DNA. A DNA replisome is responsible for the rapid and accurate replication of genomic DNA. The phage T4 holoenzyme (HE) is composed of the gp43 polymerase and the gp45 sliding clamp. The gp45 homotrimeric ring clamp increases the processivity of gp43. It has one of its subunit interfaces partially open in solution (1, 2) and is loaded onto DNA by an ATP-dependent gp44/ 62 clamp loader. The intersubunit ATP binding sites of gp44 subunits are well suited for intersubunit communication on ATP binding and hydrolysis as revealed in a recent crystal structure of the gp45−gp44/62−DNA complex (3, 4). The ATP bound form of gp44/62 binds and opens the clamp further to facilitate its loading onto the DNA (5). The clamp interacts with gp43 recruited to the DNA primer-template (P-T) junction to form an HE (6, 7).The sequence of events that leads to gp45 loading onto DNA by gp44/62, which includes ATP consumption and stoichiometry, dynamics of ring opening and closing, and possible pathways of assembly, has been studied in detail (7-16). Here, we examine gp45 loading on DNA using a FRET-based assay involving a Cy5-gp45 and a Cy3-DNA substrate. This assay directly monitors gp45 loading onto DNA. Previous studies have examined elements of this mechanism by following the clamp ring opening and closing through distance changes derived from intersubunit FRET signals (11,17). By using the FRET between DNA and clamp, we examined the ensemble kinetics of gp45 loading onto DNA mediated by gp44/62 and then the HE assembly. Experiments with ATP vs. ATPγS revealed the timing of gp44/62 departure. We carried out complementary studies at the singlemolecule level and observed a series of FRET states stemming from intermediates involved in gp45 loading onto DNA, whose rates of interconversion simulate the en...

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Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies

Cited by 25 publications

References 42 publications

Polymerase Chaperoning and Multiple ATPase Sites Enable the E. coli DNA Polymerase III Holoenzyme to Rapidly Form Initiation Complexes

Polymerase Chaperoning and Multiple ATPase Sites Enable the E. coli DNA Polymerase III Holoenzyme to Rapidly Form Initiation Complexes

Kinetic analysis of PCNA clamp binding and release in the clamp loading reaction catalyzed by Saccharomyces cerevisiae replication factor C

How a holoenzyme for DNA replication is formed

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