Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair

Saro, Francisco J. López de

doi:10.1093/emboj/cdg603

Cited by 112 publications

(139 citation statements)

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“…It appears that the clamp may serve as a platform for multiple protein bindings and thus becomes the major bridging point between the replisome and other proteins or complexes. The clamp may orchestrate multiprotein reactions through the sequential binding of proteins to the clamp in a competitive and temporally regulated manner, as suggested by the interactions of several E. coli polymerases with the ␤-clamp (26). On the other hand, concurrent binding of several proteins to the clamp could happen if allowed sterically, as in a recently proposed ''tool-belt'' model for the pivotal role of the clamp during translesion synthesis (27).…”

Section: The Effect Of Trap Concentration At Constant Wt͞trap Polymerasementioning

confidence: 99%

The dynamic processivity of the T4 DNA polymerase during replication

Yang

Zhuang

Roccasecca

et al. 2004

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

124

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The polymerase (gp43) processivity during T4 replisome mediated DNA replication has been investigated. The size of the Okazaki fragments remains constant over a wide range of polymerase concentrations. A dissociation rate constant of Ϸ0.0013 sec ؊1 was measured for the polymerases from both strands, consistent with highly processive replication on both the leading and lagging strands. This processive replication, however, can be disrupted by a catalytically inactive mutant D408N gp43 that retains normal affinity for DNA and the clamp. The inhibition kinetics fit well to an active exchange model in which the mutant polymerase (the polymerase trap) displaces the replicating polymerase. This kinetic model was further strengthened by the observation that the sizes of both the Okazaki fragments and the extension products on a primed M13mp18 template were reduced in the presence of the mutant polymerase. The effects of the trap polymerase therefore suggest a dynamic processivity of the polymerase during replication, namely, a solution͞replisome polymerase exchange takes place without affecting continued DNA synthesis. This process mimics the polymerase switching recently suggested during the translesion DNA synthesis, implies the multiple functions of the clamp in replication, and may play a potential role in overcoming the replication barriers by the T4 replisome.DNA replication ͉ polymerase processivity ͉ polymerase exchange B acteriophage T4 DNA polymerase is responsible for DNA synthesis on both leading and lagging strands. This enzyme is the gene 43 product (gp43), which, along with seven other T4 replication proteins, constitutes the T4 replisome that carries out coordinated DNA synthesis (1). Among these seven proteins, the clamp loader (gp44͞62) and the clamp protein (gp45) are polymerase accessory factors that significantly increase the processivity of gp43 during replication by forming the holoenzyme complex (2). The current working model of T4 DNA replication involves two such holoenzyme complexes acting on leading and lagging strands (3). Moving ahead of the holoenzyme complexes is the T4 primosome generated from the helicase (gp41), the primase (gp61), and the helicase accessory protein (gp59). This unit is required to rapidly unwind the double-stranded DNA in front of the moving fork (4, 5) and to synthesize the pentaribonucleotide primers for Okazaki fragment synthesis (6). The last replisome component is the singlestranded DNA (ssDNA) binding protein (gp32) which is involved in the stabilization of the ssDNA loop structure generated during lagging strand synthesis (7) and in the organization of the replisome (8, 9).The entire 172-kb T4 genome is fully duplicated within Ϸ15 min (10). As a result, the high processivity of the polymerase is crucial for efficient DNA replication. The holoenzyme stability has been measured on a short, defined DNA fork to give a half-life of Ϸ6 min (11). This value is within the same range as the 11-min half-life for the T4 helicase on a moving fork determined by Alberts and...

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Section: The Effect Of Trap Concentration At Constant Wt͞trap Polymerasementioning

confidence: 99%

The dynamic processivity of the T4 DNA polymerase during replication

Yang

Zhuang

Roccasecca

et al. 2004

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

124

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“…The C-terminal Residues of PolC Interact with E. coli ␤-The C-terminal 20 residues of E. coli ␣ form an essential contact with the E. coli ␤ clamp (18,58,65,66). Our previous analysis of this sequence indicates that the Gln, Leu, and Phe side chains within the last 7 residues are important contributors to the strength of the interaction with ␤ (66).…”

Section: Fig 6 S Aureus Polc Can Utilize Heterologous ␤ Clampsmentioning

confidence: 99%

Conserved Interactions in the Staphylococcus aureus DNA PolC Chromosome Replication Machine

Bruck

Georgescu

O'Donnell

2005

Journal of Biological Chemistry

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The PolC holoenzyme replicase of the Gram-positive Staphylococcus aureus pathogen has been reconstituted from pure subunits. We compared individual S. aureus replicase subunits with subunits from the Gram-negative Escherichia coli polymerase III holoenzyme for activity and interchangeability. The central organizing subunit, , is smaller than its Gram-negative homolog, yet retains the ability to bind single-stranded DNA and contains DNA-stimulated ATPase activity comparable with E. coli . S. aureus also stimulates PolC, although they do not form as stabile a complex as E. coli polymerase III⅐. We demonstrate that the extreme C-terminal residues of PolC bind to and function with ␤ clamps from different bacteria. Hence, this polymerase-clamp interaction is highly conserved. Additionally, the S. aureus ␦ wrench of the clamp loader binds to E. coli ␤. The S. aureus clamp loader is even capable of loading E. coli and Streptococcus pyogenes ␤ clamps onto DNA. Interestingly, S. aureus PolC lacks functionality with heterologous ␤ clamps when they are loaded onto DNA by the S. aureus clamp loader, suggesting that the S. aureus clamp loader may have difficulty ejecting from heterologous clamps. Nevertheless, these overall findings underscore the conservation in structure and function of Gram-positive and Gram-negative replicases despite >1 billion years of evolutionary distance between them.The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is extremely rapid (ϳ1 kb/s) and extends DNA by thousands of nucleotides without dissociating from the DNA template (1-3). These special features require accessory proteins, as the polymerase subunit alone is only weakly active in synthesis and lacks high processivity. The accessory proteins act as a clamp loader ATPase complex and a ring-shaped subunit that functions as a DNA sliding clamp (␤). The ␤ clamp encircles the DNA duplex and binds the polymerase, tethering it to DNA for rapid and processive chain extension. The clamp loader uses energy derived from ATP hydrolysis to pry open the ␤ clamp and to close it around a primed template.The E. coli ␥/ clamp loader consists of five proteins required for clamp loading ( 2 ␥ 1 ␦ 1 ␦Ј 1 ) and also the attached and ancillary subunits (4 -7). In E. coli, the / subunits connect the replicase to single-stranded DNA-binding protein (SSB), 1 but are not required for clamp loading (8 -10). The / subunits also contribute to the stability of the ␥/ complex in vitro (11). In E. coli, the dnaX gene produces two polypeptides, and ␥.(71 kDa) is the full-length product of the gene, whereas ␥ (47 kDa) is a truncated version of , produced by a translational frameshift (12)(13)(14). Both and ␥ are capable of functioning with ␦ and ␦Ј in clamp loading action (15). However, the unique C-terminal 24-kDa C-terminal region of provides extra functions relative to ␥. For example, (but not ␥) binds to the polymerase directly (16). Hence, the presence of multiple subunits within the clamp loader enables it to cross-link two polymerases, ther...

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“…Recent work suggests that the Escherichia coli sliding clamp can bind a large number of replication proteins, including all five bacterial polymerases, the clamp loader, and DNA ligase (8). All of these interactions use the same site on the clamp, and many, although not all, involve the conserved C-terminal peptide mentioned earlier.…”

Section: Biological Implicationsmentioning

confidence: 99%

T4 replication: What does “processivity” really mean?

Joyce¹

2004

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

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Competitive processivity-clamp usage by DNA polymerases during DNA replication and repair

Cited by 112 publications

References 104 publications

The dynamic processivity of the T4 DNA polymerase during replication

The dynamic processivity of the T4 DNA polymerase during replication

Conserved Interactions in the Staphylococcus aureus DNA PolC Chromosome Replication Machine

T4 replication: What does “processivity” really mean?

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