Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

Abstract:

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Kinetic Parameters, Nucleotide Analogs / Template Lesions, Accessory Proteins/Complexes, Nucleotide Incorporation, Exonuclease Activity, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Eco Pol III Bonner CA1992 3-5' Exonuclease (proofreading) Yes
Eco Pol III Bonner CA1992 Full length or truncated Full length
Eco Pol III Bonner CA1992 Processivity 5000bp
Eco Pol III Bonner CA1992 Nucleotide incorporation accessory protein(s) Beta and gamma complex form beta-sliding clamp
Eco Pol I Bonner CA1992 Molecular Weight 1.03E+05 Dalton
Eco Pol I Bonner CA1992 3-5' Exonuclease (proofreading) Yes
Eco Pol I Bonner CA1992 Cloned or native Native organism
Eco Pol I Bonner CA1992 5-3' Exonuclease Yes
Eco Pol I Bonner CA1992 Template lesions Bypasses Reaction: Nucleotide incorporation; DNA lesion: Apurinic/Apyrimidinic (AP) site
Eco Pol I Bonner CA1992 Full length or truncated Full length
Eco Pol II Bonner CA1992 Molecular Weight 8.99E+04 Dalton
Eco Pol II Bonner CA1992 3-5' Exonuclease (proofreading) Yes
Eco Pol II Bonner CA1992 Cloned or native Native organism
Eco Pol II Bonner CA1992 Full length or truncated Full length
Eco Pol II Bonner CA1992 Processivity 1.6kb
Eco Pol II Bonner CA1992 Vmax 25 /second Reaction: Nucleotide incorporation; Substrate: dNTPs
Eco Pol II Bonner CA1992 Nucleotide incorporation accessory protein(s) Beta and gamma subunits of Pol III

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