Characterization of DNA metabolizing enzymes in situ following polyacrylamide gel electrophoresis

Journal Article · Tue Mar 12 00:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00224a014· OSTI ID:5026407

Longley, M J; Mosbaugh, D W ^[1]

---

1. Oregon State Univ., Corvallis (United States)

The authors have detected the in situ activities of DNA glycosylase, endonuclease, exonuclease, DNA polymerase, and DNA ligase using a novel polyacrylamide activity gel electrophoresis procedure. DNA metabolizing enzymes were resolved through either native or SDS-polyacrylamide gels containing defined {sup 32}P-labeled oligonucleotides annealed to M13 DNA. After electrophoresis, these enzymes catalyzed in situ reactions and their ({sup 32}P)DNA products were resolved from the gel by a second dimension of electrophoresis through a denaturing DNA sequencing gel. Detection of modified (degraded or elongated) oligonucleotide chains was used to locate various enzyme activities. The catalytic and physical properties of Novikoff hepatoma DNA polymerase {beta} were found to be similar under both in vitro and in situ conditions. With 3{prime}-terminally matched and mismatched ({sup 32}P)DNA substrates in the same activity gel, DNA polymerase and/or 3{prime} to 5{prime} exonuclease activities of Escherichia coli DNA polymerase I (large fragment), DNA polymerase III (holoenzyme), and exonuclease III were detected and characterized. Several restriction endonucleases and the tripeptide (Lys-Try-Lys), which acts as an apurinic/apyrimidinic endonuclease, were able to diffuse into gels and modify DNA. This ability to create intermediate substrates within activity gels could prove extremely useful in delineating the steps of DNA replication and repair pathways.

• OSTI ID: 5026407
• Journal Information: Biochemistry; (United States), Vol. 30:10; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA
ELECTROPHORESIS
DNA POLYMERASES
BIOCHEMICAL REACTION KINETICS
ENDONUCLEASES
GLYCOSYL HYDROLASES
DNA SEQUENCING
LIGASES
PHOSPHORUS 32
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
DNA-ASE
ENZYMES
ESTERASES
HYDROLASES
ISOTOPES
KINETICS
LIGHT NUCLEI
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
REACTION KINETICS
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques