Processivity of the Gene 41 DNA Helicase at the Bacteriophage T4 DNA Replication Fork

Schrock, Robert D.; Alberts, Bruce

doi:10.1074/jbc.271.28.16678

Cited by 32 publications

(37 citation statements)

References 27 publications

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“…A value of Ϸ0.0013 sec Ϫ1 was obtained for both polymerases on the minicircle and the TRFII substrate. This rate constant agrees with the one (0.002 sec Ϫ1 ) measured on a static fork substrate (11) and is also close to the rate (0.001 sec Ϫ1 ) measured for the helicase during replication (12). This high processivity is further substantiated by the observation that the size of the Okazaki fragments does not increase with decreasing polymerase concentrations because diluting a dissociative replisome component would result in the formation of longer Okazaki fragments (13).…”

Section: The Effect Of Trap Concentration At Constant Wt͞trap Polymerasesupporting

confidence: 76%

“…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 coworkers (12). Dissociation halflives of this magnitude suggest that both the helicase and the polymerase within the replisome could potentially finish replicating the entire genome before dissociation.…”

mentioning

confidence: 57%

“…where y is the amount of dNMP incorporation, F 0 is the initial fork number, R is the fork rate (150 nucleotides per sec), and t is the time after dilution (12). The k off measured for both strands on either the minicircle (Ϸ0.0013 sec Ϫ1 ) or the TRFII substrate (Ϸ0.0017 sec Ϫ1 , data not shown) are in good agreement and are also consistent with the holoenzyme off rate measured earlier on a small fork substrate (11), suggesting that both holoenzymes already possess an intrinsic processivity that is not further enhanced by the presence of primosome proteins.…”

Section: Measurement Of Polymerase Dissociation Rate By Dilutionmentioning

confidence: 99%

“…during active replication. On both the minicircle and TRFII substrates, a dilution experiment was designed to measure the processivity of the replisome polymerases, similar to one devised for evaluating the processivity of the helicase during replication (12). The replisomes were assembled, and replication was initiated.…”

Section: Measurement Of Polymerase Dissociation Rate By Dilutionmentioning

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 Polymerasesupporting

confidence: 76%

mentioning

confidence: 57%

Section: Measurement Of Polymerase Dissociation Rate By Dilutionmentioning

confidence: 99%

Section: Measurement Of Polymerase Dissociation Rate By Dilutionmentioning

confidence: 99%

See 2 more Smart Citations

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

View full text Add to dashboard Cite

show abstract

“…The simplest is to determine the sensitivity of DNA synthesis to dilution of individual components. This approach has demonstrated that the helicase (gp41) is very stably associated with the replication fork (1). By contrast, lagging strand synthesis was sensitive to dilution of the clamp (gp45), clamp loader (gp44͞62 complex), and primase (gp61) (5,6), reflecting the remodeling of the lagging strand machinery that must take place as each new Okazaki fragment is started.…”

Section: Processivity Measurementsmentioning

confidence: 99%