Building a Replisome from Interacting Pieces

Shamoo, Yousif; Steitz, Thomas A.

doi:10.1016/s0092-8674(00)81647-5

Cited by 352 publications

(215 citation statements)

References 52 publications

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“…3B) indicate that an interaction between the incoming polymerase and the clamp has to be established in order for the former to displace the replicating polymerase. A model of gp45 clamp͞RB69 polymerase (a close relative of T4 gp43) interaction has been built based on crystal structure information (19). The polymerase C-terminal region was shown to interact with the interdomain loop of one of the clamp subunits.…”

Section: The Effect Of Trap Concentration At Constant Wt͞trap Polymerasementioning

confidence: 99%

“…However, solution studies revealed a clamp structure with one open and two closed interfaces (20) and strongly argued for an interaction between the polymerase C terminus and the open clamp subunit interface (21,22). The x-ray data and its associated holoenzyme model which used the weaker interdomain binding site of gp45 was predicted, however, to play some role in holoenzyme assembly, DNA replication, translesion bypass, or other replication associated processes (19,21).…”

Section: The Effect Of Trap Concentration At Constant Wt͞trap Polymerasementioning

confidence: 99%

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The dynamic processivity of the T4 DNA polymerase during replication

Yang

Zhuang

Roccasecca

et al. 2004

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

122

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%

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.

122

124

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“…The oligonucleotide (1 pmol) was first labeled at its 5Ј terminus with 33 P by incubation in a reaction mixture (20 l) containing 70 mM Tris-Cl, pH 7.5, 10 mM MgCl 2 , 5 mM DTT, 5 l of [␥-33 P]ATP (2500 Ci/mmol), and 1 l of T4 polynucleotide kinase at 37°C for 30 min, and then annealed to M13 DNA. The 5Ј-33 P-labeled, 100-nt ssDNA substrate used for processivity measurements on ssDNA was prepared by alkali denaturation of the dsDNA substrate by treatment with 50 mM NaOH at 20°C for 15 min, followed by neutralization with HCl.…”

Section: Methodsmentioning

confidence: 99%

“…Yet, the primer terminus needs to traverse the ϳ35 Å that separate the polymerase and exonuclease active sites. This switch is effected by conformational changes in the polymerase as well as in the orientation of the helical axis of DNA, such as those observed in the crystal structure of RB69 DNA polymerase in the editing mode (33). The fact that the exonuclease activity on dsDNA is lowered even more than on ssDNA raises the possibility that T7 DNA polymerase undergoes a conformational change during the proofreading reaction and that residues 144 -157 may play a role during the postulated conformational change.…”

Section: Figmentioning

confidence: 99%

A Unique Region in Bacteriophage T7 DNA Polymerase Important for Exonucleolytic Hydrolysis of DNA

Kumar

Chiu

Tabor

et al. 2004

Journal of Biological Chemistry

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A crystal structure of the bacteriophage T7 gene 5 protein/Escherichia coli thioredoxin complex reveals a region in the exonuclease domain (residues 144 -157) that is not present in other members of the E. coli DNA polymerase I family. To examine the role of this region, a genetically altered enzyme that lacked residues 144 -157 (T7 polymerase (pol) ⌬144 -157) was purified and characterized biochemically. The polymerase activity and processivity of T7 pol ⌬144 -157 on primed M13 DNA are similar to that of wild-type T7 DNA polymerase implying that these residues are not important for DNA synthesis. The ability of T7 pol ⌬144 -157 to catalyze the hydrolysis of a phosphodiester bond, as judged from the rate of hydrolysis of a p-nitrophenyl ester of thymidine monophosphate, also remains unaffected. However, the 3 -5 exonuclease activity on polynucleotide substrates is drastically reduced; exonuclease activity on single-stranded DNA is 10-fold lower and that on double-stranded DNA is 20-fold lower as compared with wild-type T7 DNA polymerase. Taken together, our results suggest that residues 144 -157 of gene 5 protein, although not crucial for polymerase activity, are important for DNA binding during hydrolysis of polynucleotides.Bacteriophage T7-encoded gene 5 protein is a replicative DNA polymerase that also has an associated 3Ј-5Ј exonuclease activity that is active on both single-stranded (ss) 1 and doublestranded (ds) DNA (1). By itself, the 80-kDa gene 5 protein (gp5) is distributive for DNA synthesis and catalyzes the addition of less than 15 nucleotides before dissociating from a primer-template terminus (2). Association in a 1:1 complex with the 12-kDa Escherichia coli host protein thioredoxin increases the processivity of polymerization by gp5; the gp5⅐thioredoxin complex catalyzes the addition of thousands of nucleotides per cycle of polymerization at a rate of 300 nucleotides per second (3, 4). The association with thioredoxin also increases the exonuclease activity of gp5 on dsDNA several hundred-fold but has little effect on the hydrolysis of ssDNA (2). The complex of gp5 and thioredoxin will be referred to as T7 DNA polymerase in this report.T7 DNA polymerase, like other members of the E. coli DNA polymerase I (Pol I) family, has a bipartite architecture with distinct C-terminal polymerase and N-terminal exonuclease domains (5). A 2.2-Å resolution crystal structure of T7 DNA polymerase in complex with a primer-template and an incoming dideoxynucleoside triphosphate was solved in the polymerization mode (see Fig. 1). This structure captured the polymerase in the act of adding a nucleotide to a growing chain of DNA. The polymerase domain is likened to a right hand composed of thumb, fingers, and palm subdomains that converge to form a DNA binding groove that leads to the polymerase active site. The processivity factor, E. coli thioredoxin, is bound to the thumb subdomain. In this structure, there are no visible contacts between the exonuclease domain and DNA. The exonuclease and polymerase active sites a...

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“…If the bypass polymerase has a lower intrinsic affinity for the primer terminus than does the replicative polymerase, this scenario would facilitate exchange subsequent to lesion bypass, thus avoiding the synthesis of long tracts of DNA by the less accurate bypass polymerase. It also has been suggested that transient release of DNA by a replicating polymerase could relieve torsional stress at the replication fork and could facilitate transfer of a mispaired primer terminus to the editing site (9,12).…”

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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Building a Replisome from Interacting Pieces

Cited by 352 publications

References 52 publications

The dynamic processivity of the T4 DNA polymerase during replication

The dynamic processivity of the T4 DNA polymerase during replication

A Unique Region in Bacteriophage T7 DNA Polymerase Important for Exonucleolytic Hydrolysis of DNA

T4 replication: What does “processivity” really mean?

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