TY - JOUR
T1 - Identification of a transient excision intermediate at the crossroads between DNA polymerase extension and proofreading pathways
AU - Baker, Rosanna P.
AU - Reha-Krantz, Linda J.
PY - 1998/3/31
Y1 - 1998/3/31
N2 - DNA polymerases achieve accurate DNA replication through a delicate balance between primer elongation and proofreading. While insufficient proofreading results in DNA replication errors, indiscriminate removal of correct along with incorrect nucleotides is wasteful and may prevent completion of DNA synthesis. The transition between polymerization and proofreading modes is proposed to be governed by a kinetic barrier that prevents proofreading unless the rate of primer elongation is significantly reduced by the presence of an incorrect base pair at the primer-terminus. We have used mutational analysis, coupled with a sensitive, fluorescence-based assay to characterize intermediate steps in the proofreading pathway. A highly fluorescent complex forms between the bacteriophage T4 DNA polymerase and DNA primer-templates labeled at the 3' terminus with the base analog 2- aminopurine. Formation of the fluorescent complex appears to be a rate- determining step in the proofreading pathway and is impaired for several mutator T4 DNA polymerases with amino acid substitutions in the exonuclease domain. Although these mutant DNA polymerases are proficient in hydrolysis, their reduced ability to form the fluorescent complex imposes a higher kinetic barrier. As a consequence, the mutant DNA polymerases proofread less frequently, resulting in more DNA replication errors.
AB - DNA polymerases achieve accurate DNA replication through a delicate balance between primer elongation and proofreading. While insufficient proofreading results in DNA replication errors, indiscriminate removal of correct along with incorrect nucleotides is wasteful and may prevent completion of DNA synthesis. The transition between polymerization and proofreading modes is proposed to be governed by a kinetic barrier that prevents proofreading unless the rate of primer elongation is significantly reduced by the presence of an incorrect base pair at the primer-terminus. We have used mutational analysis, coupled with a sensitive, fluorescence-based assay to characterize intermediate steps in the proofreading pathway. A highly fluorescent complex forms between the bacteriophage T4 DNA polymerase and DNA primer-templates labeled at the 3' terminus with the base analog 2- aminopurine. Formation of the fluorescent complex appears to be a rate- determining step in the proofreading pathway and is impaired for several mutator T4 DNA polymerases with amino acid substitutions in the exonuclease domain. Although these mutant DNA polymerases are proficient in hydrolysis, their reduced ability to form the fluorescent complex imposes a higher kinetic barrier. As a consequence, the mutant DNA polymerases proofread less frequently, resulting in more DNA replication errors.
UR - http://www.scopus.com/inward/record.url?scp=0032584187&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032584187&partnerID=8YFLogxK
U2 - 10.1073/pnas.95.7.3507
DO - 10.1073/pnas.95.7.3507
M3 - Article
C2 - 9520396
AN - SCOPUS:0032584187
SN - 0027-8424
VL - 95
SP - 3507
EP - 3512
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 7
ER -