Chaperoning of a Replicative Polymerase onto a Newly Assembled DNA-Bound Sliding Clamp by the Clamp Loader

Downey, Christopher D.; McHenry, Charles S.

doi:10.1016/j.molcel.2010.01.013

Cited by 43 publications

(62 citation statements)

References 45 publications

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“…ATP␥S, which is not hydrolyzed by the DnaX complex, supports the initiation complex formation provided that the form of DnaX is present (4,5). Recently, we have demonstrated that 3 complexes chaperone the associated pol III onto a newly loaded ␤ 2 with the kinetics and affinity required to support rapid sequential Okazaki fragment synthesis (5). That study supported a model in which pol III, if bound to , could participate in initiation complex formation in the presence of ATP␥S by attacking transiently loaded ␤ 2 and driving initiation complex formation to completion.…”

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

confidence: 80%

“…This difference in effects is consistent with other observations that ATP binding is more important than ATP hydrolysis in initiation complex formation. ATP␥S, which is not hydrolyzed by the DnaX complex, supports the initiation complex formation provided that the form of DnaX is present (4,5). Recently, we have demonstrated that 3 complexes chaperone the associated pol III onto a newly loaded ␤ 2 with the kinetics and affinity required to support rapid sequential Okazaki fragment synthesis (5).…”

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

confidence: 86%

“…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 analog, ATP␥S (4). In addition, -containing DnaX complexes chaperone pol III onto the newly loaded ␤ 2 , reducing the required levels of pol III and enabling initiation rates sufficient to support Okazaki fragment synthesis under physiological conditions (5). This chaperoning reaction is required for ATP␥S-dependent initiation complex formation.…”

Section: Dna Polymerase III Holoenzyme (Pol Iii He)mentioning

confidence: 99%

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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 Prommentioning

confidence: 80%

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

confidence: 86%

Section: Dna Polymerase III Holoenzyme (Pol Iii He)mentioning

confidence: 99%

See 1 more Smart Citation

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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“…This binding activity would create a hierarchy for access to the primer terminus, a view of the toolbelt model in which clamp-polymerase interactions do more than merely increase the local polymerase concentration at the DNA template. During normal growth conditions, Pol III, which is preferentially loaded to the primer terminus (38), performs the majority of DNA synthesis. Pol IV is able to simultaneously bind β in an inactive mode, although it is currently unclear how other β-interacting proteins would influence its occupancy in vivo.…”

Section: Discussionmentioning

confidence: 99%

Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis

Kath

Jergic

Heltzel

et al. 2014

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

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Translesion synthesis (TLS) by Y-family DNA polymerases alleviates replication stalling at DNA damage. Ring-shaped processivity clamps play a critical but ill-defined role in mediating exchange between Y-family and replicative polymerases during TLS. By reconstituting TLS at the single-molecule level, we show that the Escherichia coli β clamp can simultaneously bind the replicative polymerase (Pol) III and the conserved Y-family Pol IV, enabling exchange of the two polymerases and rapid bypass of a Pol IV cognate lesion. Furthermore, we find that a secondary contact between Pol IV and β limits Pol IV synthesis under normal conditions but facilitates Pol III displacement from the primer terminus following Pol IV induction during the SOS DNA damage response. These results support a role for secondary polymerase clamp interactions in regulating exchange and establishing a polymerase hierarchy.single-molecule techniques | DNA replication | DNA repair | lesion bypass | DinB

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“…Polymerase recycling, a key element in this elegant model, has been validated by several landmark biochemical studies: 1) lagging-strand synthesis is resistant to polymerase dilution in ensemble studies and in single-molecule investigations (22,23); 2) OF size distribution is independent of polymerase concentration (17,24); and 3) leading-and lagging-strand polymerases are all bound within a replisome via protein-protein interactions (25,26).…”

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confidence: 99%

Insights into Okazaki Fragment Synthesis by the T4 Replisome

Chen

Yue

Spiering

et al. 2013

Journal of Biological Chemistry

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Background:The T4 replisome duplicates the lagging DNA strand through discontinuous Okazaki fragments. Results: A third polymerase is not involved in repetitive Okazaki fragment synthesis. Conclusion: The T4 replisome recycles the lagging-strand polymerase but looses the clamp in each Okazaki fragment cycle through a collision or signaling pathway. Significance: Elucidation of the behavior of polymerase, clamp and clamp loader for Okazaki fragment synthesis broadens our understanding of coordinated DNA replication.

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Chaperoning of a Replicative Polymerase onto a Newly Assembled DNA-Bound Sliding Clamp by the Clamp Loader

Cited by 43 publications

References 45 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

Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis

Insights into Okazaki Fragment Synthesis by the T4 Replisome

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