Repair of depurinated DNA in vitro by enzymes purified from human lymphoblasts.

Abstract

Alkali-labile lesions introduced into 17 DNA by treatment with methyl methanesulfonate were removed and the DNA was repaired by incubation with DNA polymerase a and nuclease from a human lymphoblastoid line followed by the addition of DNA ligase. The nuclease preparation contains both arurinic endonuclease and 5'-3' exonuclease activities.Dinucleotides appear to be the first product of exonuclease action. Re air of methyl methanesulfonate-induced damage can occur by the insertion of only a few nucleotides per lesion as in vivo.Two different pathways for DNA excision repair have been reported in mammalian cells. Nucleotide excision repair (1), e.g., of UV-induced damage, results in the insertion of "patches" of about 100 nucleotides in human cells (2). In contrast, repair of methyl methanesulfonate (MeMes)-induced damage by the apurinic excision repair pathway results in patches of three to four nucleotides (2,3). The reason for this difference in patch size is not known, but it seems reasonable to suppose that the different pathways use enzymes with different processive character (4). To study the factors involved in determining patch size, we are attempting to reconstruct repair systems in vitro that duplicate the in vivo characteristics of a particular repair pathway. In this paper we describe a system that carries out small patch apurinic repair using nuclease and polymerase activities derived from a human lymphoma cell line. MATERIALS AND METHODSCell Culture. The human Burkitt's lymphoma line Daudi (5), obtained from a biopsy and provided by G. Klein, was adapted to spinner culture and grown in RPMI 1640 medium with 16% fetal calf serum (GIBCO) to a cell density of 1-1.5 X 106/ml. Cells were collected, washed, and stored at -700.Partial Purification of DNA Polymerase a and Nuclease Activity. Daudi cells (100 g) in 20 mM potassium phosphate, pH 7.4/10 mM 2-mercaptoethanol/20% glycerol (buffer A; all phosphate buffers were the potassium salt at pH 7.4; all contain mercaptoethanol and glycerol unless so noted) were disrupted in a nitrogen bomb. The cell supernatant was precipitated with ammonium sulfate and the precipitate (30-75% saturation) was dialyzed against buffer A and applied to a 2.5 X 26-cm DEAE-cellulose column (DE52) to remove nucleic acid. The column was equilibrated and eluted with 0.2 M phosphate. The protein peak fractions were dialyzed against buffer A, applied to a hydroxylapatite column [2.5 X 13.5 cm, Bio-Gel HTP (BioRad)], and eluted with a linear 0.02-0.5 M phosphate gradient. Pooled fractions containing polymerase activity were concentrated with polyethylene glycol 6000 and chromatographed on Sephadex G-100 (1.5 X 110 cm) with 0.1 M phosphate. The DNA polymerase peak fractions were combined, dialyzed (50 mM phosphate), applied to a small (6-ml bed volume) hydroxylapatite column, and eluted with 0.05-0.3 M phosphate. Pooled peak fractions were applied to 6 ml of DNA-agarose (6) equilibrated with 10 mM phosphate/20 mM KCI/10 mM 2-mercaptoethanol/10% glycerol and eluted with a linea...

Enzymatic insertion of purine bases into depurinated DNA in vitro.

“…Subsequently, excision, polymerization, and ligation steps occur, leading to restoration of the DNA integrity. Endonucleases specific for apurinic sites have been purified from various sources (5,(8)(9)(10)(11)(12)(13), and the total repair of apurinic sites in DNA in vitro has been demonstrated with bacterial (14) and human enzymes (15).…”

Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases.

“…T4 DNA polymerase was a gift from Nicholas Cozzarelli, pol III was a gift from U. Hubscher, and avian myeloblastosis virus (AMV) reverse transcriptase was provided by J. W. Beard. DNA polymerase a from Daudi human lymphoma cells was prepared as described (10) (52 units), the reaction mixture also contained 5 mM ATP and 5 mM spermidine HCl. The amounts of polymerases used were chosen to give maximum incorporation of label.…”

Abasic sites from cytosine as termination signals for DNA synthesis

“…AMV reverse transcriptase was from Life Sciences. DNA polymerase a was prepared by Dr. K. Bose from the human lymphoma line Daudi (9). Polymerase 3 from Novikoff hepatoma, was purified by Mosbaugh and Meyer and obtained from Dr. T. Kunkel.…”