Excess beta subunit can bypass the ATP requirement for highly processive synthesis by the Escherichia coli DNA polymerase III holoenzyme.

Crute, James J.; LaDuca, R J; Johanson, Kyung; McHenry, Charles S.; Bambara, Robert A.

doi:10.1016/s0021-9258(17)44423-1

Cited by 40 publications

(5 citation statements)

References 15 publications

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“…Previous work on the E. coli DNA polymerase III holoenzyme from the Bambara laboratory (Crute et al, 1983) demonstrated that very large amounts of protein can bypass to some extent the requirement for ATP and other accessory proteins of the E. coli DNA polymerase III holoenzyme. Unpublished observations from our laboratory (M. K. Reddy, S. E. Weitzel, and P. H. von Hippel, in preparation) also suggest that under certain conditions the addition of large amounts of gp45 alone to defined primer-template substrates (30/50-mer) results in an increase in processivity of gp43, both in its exonuclease and in its synthesis mode.…”

Section: Holoenzyme Assembly: the Four-and Five-protein Systemsmentioning

confidence: 99%

Structure and function of the bacteriophage T4 DNA polymerase holoenzyme

Young¹,

Reddy²,

Hippel³

1992

Biochemistry

108

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Section: Holoenzyme Assembly: the Four-and Five-protein Systemsmentioning

confidence: 99%

Structure and function of the bacteriophage T4 DNA polymerase holoenzyme

Young¹,

Reddy²,

Hippel³

1992

Biochemistry

108

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“…Even though this complex is not as stable to 1 Abbreviations: DMSM, 2,5-dimethoxysti)bene-4-maleimide; FM, fluorescein-5-maleimide; HEPES, A'-(2-hydroxyethyl)piperazine-,V'-2ethanesulfonic acid; holoenzyme, DNA polymerase III holoenzyme; PM, ¿V-1-pyrenylmaleimide; pol III*, DNA polymerase III holoenzyme subassembly lacking the ß subunit. gel filtration (Crute et al, 1983), a nondisruptive technique has shown that this initiation complex is nearly as stable as that formed with ATP (Kwon-Shin et al, 1987). When a deoxynucleoside triphosphate mixture is added to this complex, highly processive replication occurs without the lag observed in the presence of ATP.…”

mentioning

confidence: 99%

Dimer of the .beta. subunit of Escherichia coli DNA polymerase III holoenzyme is dissociated into monomers upon binding magnesium(II)

Griep

McHenry

1988

Biochemistry

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The beta subunit of Escherichia coli DNA polymerase III holoenzyme binds Mg2+. Reacting beta with fluoresceinmaleimide (FM) resulted in one label per beta monomer with full retention of activity. Titration of FM-beta with Mg2+ resulted in a saturable 11% fluorescence enhancement. Analysis indicated that there was one noncooperative magnesium binding site per beta monomer with a dissociation constant of 1.7 mM. Saturable fluorescence enhancement was also observed when titration was with Ca2+ or spermidine(3+) but not with the monovalent cations Na+ and K+. The Mg2+-induced fluorescence enhancement was specific for FM-beta and was not observed with FM-glutathione, dimethoxystilbenemaleimide-beta, or pyrenylmaleimide-beta. Gel filtration studies indicated that the beta dimer-monomer dissociation occurred at physiologically significant beta concentrations and that the presence of 10 mM Mg2+ shifted the dimer-monomer equilibrium to favor monomers. Both the gel-filtered dimers and the gel-filtered monomers were active in the replication assay. These and other results suggested that the fluorescence increase which accompanies beta dissociation is due to a relief from homoquenching of FM when the beta dimer dissociates into monomers.

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“…Although DNA polymerase III holoenzyme is capable of highly processive primer elongation, it is easily dissociated by encountering the 5' ends of primers. In fact, on poly(dA)oligo(dT)10, product sizes rarely exceed twice the average gap size between primers (Crute et al, 1983). Processive primer elongation by DNA polymerases a and 8 is less sensitive to primer density at all conditions tested.…”

Section: Discussionmentioning

confidence: 92%

Calf thymus DNA polymerases .alpha. and .delta. are capable of highly processive DNA synthesis

Sabatino

Myers²,

Bambara³

et al. 1988

Biochemistry

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We have demonstrated that calf thymus DNA polymerases alpha and delta are capable of highly processive DNA synthesis. Processivity values between 300 and 2000 nucleotides were observed when poly(dA)-oligo(dT) or singly primed single-stranded circular bacteriophage M13 DNA at pH 6.0 and 1 mM magnesium chloride was used. These conditions do not correlate with conditions, pH 7.0 and 5 mM magnesium chloride, that support the maximum synthetic rate. Lowering the pH and magnesium concentration lowers the Km value of the reaction with respect to primer terminus concentration. Furthermore, under these same conditions, both polymerases become insensitive to dissociation from the template as a result of encountering the 5' ends of primers. Overall, these results suggest that the affinity of the polymerases for the primer termini is higher throughout the polymerization reaction of pH and magnesium concentrations are lowered from those favoring maximum synthetic rate. Experiments with short primer templates, however, indicate that this higher affinity does not cause the DNA polymerase to remain stably bound after synthesizing up to the end of the template.

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Excess beta subunit can bypass the ATP requirement for highly processive synthesis by the Escherichia coli DNA polymerase III holoenzyme.

Cited by 40 publications

References 15 publications

Structure and function of the bacteriophage T4 DNA polymerase holoenzyme

Structure and function of the bacteriophage T4 DNA polymerase holoenzyme

Dimer of the .beta. subunit of Escherichia coli DNA polymerase III holoenzyme is dissociated into monomers upon binding magnesium(II)

Calf thymus DNA polymerases .alpha. and .delta. are capable of highly processive DNA synthesis

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