Multiple ATP Binding Is Required to Stabilize the “Activated” (Clamp Open) Clamp Loader of the T4 DNA Replication Complex

Pietroni, Paola; Hippel, Peter H. von

doi:10.1074/jbc.m804371200

Cited by 27 publications

(35 citation statements)

References 32 publications

(49 reference statements)

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“…Our result that initiation complex formation does not require hydrolysis by each ATPase subunit is analogous to previous results for the bacteriophage T4 gp44/62 clamp loader (18). In that work, it was argued that ATPase activity observed in simple model clamp loading reactions may not reflect the true activity required for formation of authentic, full processive replication complexes.…”

Section: One Atp Binding-competent Dnax Subunit Is Sufficient To Promsupporting

confidence: 89%

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

Wieczorek

Downey

Dallmann

et al. 2010

Journal of Biological Chemistry

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The DnaX complex (DnaX 3 ␦␦) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, ␤ 2 , onto DNA. The complex contains three DnaX subunits, which occur in two forms: and the shorter ␥, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant or ␥ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATPbinding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. ␤ 2 binding (determined functionally) is diminished 12-30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for ␤ 2 . DNA synthesis activity can be restored by increased concentrations of ␤ 2 . In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances ␤ 2 loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes. DNA polymerase III holoenzyme (pol III HE)2 exhibits features common to all other cellular replicases. It is a tripartite assembly composed of a replicative polymerase (pol III), a sliding clamp processivity factor (␤ 2 ), and an AAAϩ ATPase that assembles the ␤ 2 clamp around DNA (DnaX complex) (1, 2). The DnaX complex contains three DnaX subunits and one each of ␦, ␦Ј, , and . The DnaX subunit is either the full-length dnaX translation product or a shorter version, ␥, which is generated by translational frameshifting. ␥ lacks two C-terminal domains that bind the replicative helicase and pol III.Current models for clamp loading, largely derived from ␥-only DnaX complexes in the absence of pol III, propose that ATP binds to all three DnaX subunits, increasing the affinity of the clamp loader for ␤ 2 and primed DNA (1, 3). Once a ␤ 2 -primed DNA complex is formed, hydrolysis of the three ATPs places the DnaX complex into a conformation with decreased affinity for DNA, leading to its dissociation from DNA-bound ␤ 2 (3). pol III then associates with ␤ 2 in a downstream step, leading to formation of an initiation complex for processive replication.We have shown that some of the characteristics of initiation complex formation catalyzed by -containing DnaX complexes are different from ␥-only complexes. For example, complex, when bound to pol III, can form initiation complexes in the leading strand half of a dimeric replicase in the presence of the nonhydrolyzed an...

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Section: One Atp Binding-competent Dnax Subunit Is Sufficient To Promsupporting

confidence: 89%

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

Wieczorek

Downey

Dallmann

et al. 2010

Journal of Biological Chemistry

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“…S2A). These observations revealed that ATP binding was necessary for gp45 loading by gp44/62 onto DNA in agreement with previous studies (7,12,18). Although the observation of a FRET in this case does not require the loaded clamp be closed around the DNA, previous work where the subunit interfaces of the clamp were labeled to create a FRET pair showed that under identical conditions the clamp would close (11, 17).…”

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confidence: 90%

“…S3 B-D). These data suggest that ATP hydrolysis may not be absolutely necessary for gp45 loading, in agreement with previous studies and those for Escherichia coli and yeast clamp loading (7,12,(18)(19)(20) We carried out pull-down experiments (data and analyses in SI Appendix, Fig. S7).…”

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confidence: 89%

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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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“…The clamp is loaded onto DNA by the clamp loader, a five-subunit complex of AAAϩ family proteins that couple ATP binding and hydrolysis to mechanical work (8 -13). Structural and mechanistic analyses of clamp loaders such as E. coli ␥ complex (11,14), bacteriophage T4 gp44/62 (8,15,16), and Saccharomyces cerevisiae RFC (10,(17)(18)(19) among others, have shown that ATP binding enables the clamp loader to bind and open the clamp and bind ptDNA, and ATP hydrolysis leads to the release of the clamp-ptDNA product (see Fig. 1A), which can then be used by DNA polymerase and other proteins.…”

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confidence: 99%

Impact of Individual Proliferating Cell Nuclear Antigen-DNA Contacts on Clamp Loading and Function on DNA

Zhou

Hingorani

2012

Journal of Biological Chemistry

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Background: Crystal structures reveal direct clamp-DNA contacts whose functions are unclear. Results: Mutations of single N-terminal arginines/lysines in the inner PCNA ring affect DNA binding-linked steps in the clamp loading mechanism. Conclusion: Loss of individual PCNA-DNA contacts disrupts clamp loading and its interaction with DNA. Significance: DNA binding to specific locations within circular clamps influences other proteins that work with clamps on DNA.

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Multiple ATP Binding Is Required to Stabilize the “Activated” (Clamp Open) Clamp Loader of the T4 DNA Replication Complex

Cited by 27 publications

References 32 publications

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

How a holoenzyme for DNA replication is formed

Impact of Individual Proliferating Cell Nuclear Antigen-DNA Contacts on Clamp Loading and Function on DNA

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