DNA-repair reactions by purified HeLa DNA polymerases and exonucleases

Journal Article · Mon Sep 05 04:00:00 EDT 1988 · J. Biol. Chem.; (United States)

OSTI ID:7095942

Randahl, H; Elliott, G C; Linn, S

PM2 duplex DNA substrates containing small gaps were utilized to study DNA repair reactions of extensively purified HeLa DNase V (a bidirectional double strand DNA exonuclease) and DNA polymerases beta, gamma (mitochondrial and extramitochondrial), and alpha holoenzyme, and delta as a function of ionic strength. At 50 mM NaCl, DNase V carried out extensive exonucleolytic degradation, and beta-polymerase exhibited strand displacement synthesis. However, at 150 mM NaCl, the DNase appeared only to remove damaged nucleotides from DNA termini while beta-polymerase catalyzed only gap-filling synthesis. When present in equimolar amounts, beta-polymerase and DNase V (which can be isolated as a 1:1 complex) catalyzed more degradation than synthesis at 50 mM NaCl; however, at 150 mM NaCl a coupled very limited nick translation reaction ensued. At physiological ionic strength DNA polymerase alpha holoenzyme was not active upon these substrates. In 15 mM KCl it could fill small gaps and carry out limited nick translation with undamaged DNA, but it could not create a ligatable substrate from UV-irradiated DNA incised with T4 UV endonuclease. Mitochondrial DNA polymerase gamma was more active at 150 mM NaCl than at lower ionic strengths. It readily filled small gaps but was only marginally capable of strand-displacement synthesis. The extramitochondrial form of gamma-polymerase, conversely, was less sensitive to ionic strength; it too easily filled small gaps but was not effective in catalyzing strand displacement synthesis. Finally, DNA polymerase delta was able to fill gaps of several to 20 nucleotides in 0.05 M NaCl, but at higher NaCl concentrations there was little activity. DNA polymerases delta did not demonstrate strand displacement synthesis.

• Research Organization: Univ. of California, Berkeley (USA)
• OSTI ID: 7095942
• Journal Information: J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 263:25; ISSN JBCHA
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
CELL CONSTITUENTS
DNA
DNA POLYMERASES
DNA REPAIR
DNA-ASE
ENDONUCLEASES
ENZYMES
ESTERASES
HELA CELLS
HYDROLASES
KINETICS
MITOCHONDRIA
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
ORGANOIDS
PHOSPHODIESTERASES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
REACTION KINETICS
RECOVERY
REPAIR
TRANSFERASES