A novel in vitro assay to study the mechanism by which DNA polymerases bypass blocking lesions to DNA replication
We devised a simple gel assay to measure insertion kinetics for any dNTP substrate opposite a target site. Our ability to synthesize an abasic lesion and place it at a single site in synthetic oligonucleotides allows for an in vitro analysis of the mechanism by which DNA polymerases bypass blocking lesions to DNA replication and to identify E. coli polymerases and accessory proteins that allow for insertion and bypass of such lesions. Using this assay we examine the preferred insertion of dATP by Drosophila DNA polymerase {alpha} opposite the abasic lesion compared to dGTP, dCTP, and dTTP for all different nearest-neighbors. The preferred insertion of dATP is governed by a V{sub max} discrimination little affected by nearest-neighbors. A DNA polymerase activity was purified from E coli, deleted for DNA polymerase I, that appears to be part of the SOS response of E. coli since it cannot be induced in lexA(Ind{sup {minus}}) strains. This inducible polymerase is DNA polymerase II. In contrast to DNA polymerase III, DNA polymerase II efficiently incorporates nucleotides opposite the abasic lesion and continues DNA synthesis. We addressed the role of E. coli DNA polymerase I targeted SOS mutagenesis.
- Research Organization:
- University of Southern California, Los Angeles, CA (USA)
- OSTI ID:
- 6829719
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
DNA POLYMERASES
BIOCHEMICAL REACTION KINETICS
ESCHERICHIA COLI
DNA REPLICATION
ATP
BIOASSAY
DROSOPHILA
ENZYME ACTIVITY
IN VITRO
ANIMALS
ARTHROPODS
BACTERIA
DIPTERA
ENZYMES
FLIES
FRUIT FLIES
INSECTS
INVERTEBRATES
KINETICS
MICROORGANISMS
NUCLEIC ACID REPLICATION
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
REACTION KINETICS
TRANSFERASES
550200* - Biochemistry