title: DNA-repair reactions by purified HeLa DNA polymerases and exonucleases.
abstract: 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. Therefore, at physiological ionic strength, it appears that either DNA polymerase beta or extramitochondrial DNA polymerase gamma might aid in short patch DNA repair of nuclear (or transfecting) DNAs, whereas mitochondrial gamma-polymerase might fill small gaps in mitochondrial DNA.
authors.affiliation: Department of Biochemistry, University of California, Berkeley 94720.
authors.forename: H
authors.initials: H
authors.lastname: Randahl
authors.forename: G C
authors.initials: GC
authors.lastname: Elliott
authors.forename: S
authors.initials: S
authors.lastname: Linn
mesh_terms: D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D011088:DNA Ligases / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004260:DNA Repair; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism*; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008928:Mitochondria / Q000201:enzymology; D009492:Neurospora crassa / Q000201:enzymology; D009994:Osmolar Concentration; D015719:Single-Strand Specific DNA and RNA Endonucleases; D012965:Sodium Chloride / Q000494:pharmacology; D013604:T-Phages / Q000201:enzymology; D014466:Ultraviolet Rays
chemical_list: D012965:Sodium Chloride; D004247:DNA; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; D004720:Endonucleases; D005090:Exodeoxyribonucleases; D005092:Exonucleases; D043211:Exodeoxyribonuclease V; D015719:Single-Strand Specific DNA and RNA Endonucleases; D011088:DNA Ligases