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Identification of a Specific Inhibitor for DNA Ligase I in Human Cells (Regulation/Replication/Repair/Recombinatlon) SHU-WEI YANG, FREDERICK F

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Identification of a Specific Inhibitor for DNA Ligase I in Human Cells (Regulation/Replication/Repair/Recombinatlon) SHU-WEI YANG, FREDERICK F Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2227-2231, March 1992 Biochemistry Identification of a specific inhibitor for DNA ligase I in human cells (regulation/repLication/repair/recombinatlon) SHU-WEI YANG, FREDERICK F. BECKER, AND JOHN Y.-H. CHAN* Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030 Communicated by Howard A. Nash, November 18, 1991 (receivedfor review October 10, 1991) ABSTRACT A protein inhibitor for human DNA ligase I DNA ligase II contained a 67-kDa polypeptide; only DNA has recently been identified. It was copurified with a fraction ligase II could utilize poly(rA)-p(dT)16 as a substrate. Phos- of the enzymes from HeLa cells through several steps of phocellulose, DEAE-agarose (Bio-Gel), CM-agarose (Bio- chromatography. The inhibitor was first identified by the Gel), hydroxylapatite, and protein standards were purchased absence of ligation activity of the associated enzyme, while it from Bio-Rad; heparin-Sepharose, Mono S HR 5/5 column, retained the ability to form the ligase-[32P]AMP adducts. The Superose 12 column, poly(dA), poly(rA), and (dT)16 were inhibitor was eluted as a single peak at -0.250.30 M NaCl from Pharmacia LKB; DNA-cellulose was from Sigma; re- from a Mono S column. It inhibited the ligation of both striction enzyme Bgl II, phage T4 DNA ligase, and T4 kinase double-stranded and single-stranded breaks by purified DNA were from GIBCO-BRL; [a-32P]ATP (3000 Ci/mmol; 1 Ci = ligase I but not by T4 DNA ligase and DNA ligase II. Subse- 37 GBq) and [y32PJATP (3000 Ci/mmol) were from ICN. quent gel-fitration chromatography indicated that this inhib- Other chemicals were from Sigma or Fisher. itor, with a molecular mass of55-75 kDa, could form a complex DNA Ligase Assays. The assay mixture (50 ,ul) for ligation with DNA ligase I and inhibited the DNA ligation activity. of single-strand DNA breaks contained 50 mM Tris-HCl (pH Rechromatography of the ligase I-inhibitor complex in high- 7.6), 10 mM MgCl2, 10 mM dithiothreitol, 1.2 mM ATP, salt conditions resulted in the dissociation of the complex and 30,000 cpm of poly(dA)4[32P]p(dT)16, and DNA ligase. After the restoration of enzyme activity, indicating that the physical incubation at 25°C for 20 min, the sample was either analyzed interaction of inhibitor with DNA ligase I is one of the mech- with gel electrophoresis or treated with calf intestine phos- anisms of inhibition. These data indicate that this protein phatase (CIP), acid-precipitated, and assayed for radioactiv- inhibitor for DNA ligase I may play a specific role in regulating ity as described (5). The ligase-AMP adduct assay and DNA ligation during replication, repair, or recombination. plasmid DNA ligation assay were carried out as described (11). DNA ligase is essential for DNA replication, repair, and Purification of the Inhibitor for DNA Ligase I from Human recombination by joining single- and double-stranded DNA Cells. All steps of the purification were carried out at 4°C. In with formation of phosphodiester bonds (1-6). Eukaryotic brief, 20 g of HeLa cells were extracted with 100 ml of DNA ligases react with ATP, forming covalent enzyme-AMP extraction buffer (0.8 M NaCl/50 mM Tris'HCI, pH 7.6/15% intermediates; then the enzymes transfer the AMP group to glycerol/l mM EDTA/10 mM 2-mercaptoethanol/10 pug of the 5' end of the DNA to complete the joining reaction (1, 2). leupeptin per ml/0.5 mM phenylmethylsulfonyl fluoride), Thus, a ligase-AMP complex assay was developed that was and the protein extraction and (NH4)2SO4 fractionations were based on the unique ability of DNA ligases to react with performed as described (11). The precipitate from (NH4)2SO4 [a-32P]ATP covalently, forming a ligase-[32P]AMP complex solution at 30-70% saturation was dissolved in and dialyzed that is sufficiently stable during SDS/polyacrylamide gel for 8 hr against buffer A (25 mM Tris HCl, pH 7.6/15% electrophoresis (SDS/PAGE) (1, 2, 4). Multiple forms of (vol/vol) glycerol/0.1 mM EDTA/10 mM dithiothreitol/0.1 DNA ligase have also been documented in mammalian cells M NaCl). The sample was then applied to a phosphocellulose (1-3, 7-14). DNA ligase I was implicated in DNA replication, column (8.5 x 2.5 cm) equilibrated with buffer A. The sample while DNA ligase II was associated with DNA repair (1-3, 10, was washed with buffer A and eluted with buffer A containing 14). However, very little is known about the regulation of 0.5 M NaCl. The eluate was diluted with buffer A without these isozymes except that earlier reports indicated that NaCl until the final NaCl concentration was 0.1 M. The poly(ADP-ribose) polymerase (PADPRP) and histones may sample was then loaded onto a DEAE-agarose column (6.0 x affect the ligation reaction nonspecifically (14-16). During 2.5 cm) preequilibrated with buffer A, and the column was the course of DNA ligases purification from HeLa cells, we washed extensively with the same buffer. The flowthroughs identified a protein inhibitor for DNA ligase I, which copu- and the washed fractions were then combined, adjusted to pH rified with a fraction of the enzyme that could form ligase- 6.5, and loaded onto a CM-agarose column (4.5 x 2.5 cm) [32P]AMP adducts. To the best of our knowledge, a DNA equilibrated with buffer B (buffer A at pH 6.5). The sample ligase I inhibitor from human cells has not been reported was washed with buffer B and then eluted with a linear previously. gradient of 0.1-0.45 M NaCl in 100 ml of buffer B. The fractions at -0.18 M NaCl were pooled, dialyzed against MATERIALS AND METHODS buffer C (10 mM potassium phosphate, pH 7.6/15% glycer- ol/10 mM dithiothreitol/0.3 M NaCI), and then loaded onto Human DNA ligases I and II were isolated from HeLa cells a hydroxylapatite column (10 x 2.0 cm). The sample was (Invitron, Berkeley, MO) as described (11), with specific washed with buffer C and eluted with a linear gradient of activities of 10,100 and 11,900 units/mg of protein, respec- 0.01-0.30 M potassium phosphate in 80 ml of buffer C. DNA tively. The ligase-AMP labeling assay indicated that DNA ligase II was eluted in a high-salt fraction (0.26 M potassium ligase I contained a 90-kDa and a 78-kDa polypeptide, while Abbreviations: PADPRP, poly(ADP-ribose) polymerase; CIP, calf The publication costs of this article were defrayed in part by page charge intestine phosphatase; E-alone, enzyme alone; E+I, enzyme with payment. This article must therefore be hereby marked "advertisement" inhibitor. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2227 Downloaded by guest on September 24, 2021 2228 Biochemistry: Yang et al. Proc. Natl. Acad. Sci. USA 89 (1992) phosphate) and was separated from DNA ligase I. The fractions eluted at about 0.2 M potassium phosphate were pooled, concentrated with a Centriprep concentrator-30 LIGATEDO (Amico), and dialyzed against buffer D (15 mM potassium LINEAR - phosphate/5 mM MgCl2/15% glycerol/10 mM dithiothrei- tol/50 mM NaCl, pH 7.6). The sample was then loaded onto a double-stranded DNA-cellulose column (5.0 x 1.0 cm) and A 8 24 26 28 30 32 34 36 38 40 4-4 4-4 _1V 4 50 52 54 5t' 5b bu washed with buffer D. The column was eluted with 15 ml of buffer D containing 0.5 M NaCl. The eluate was diluted with ;c Mc c :. buffer D without NaCl to a final concentration of 0.15 M FIG. 2. Purification of the inhibitor for DNA ligase I by Mono S NaCl, and loaded onto a heparin-Sepharose column (3.5 x column chromatography. Fractions 9 and 10 from the heparin- 1.0 cm) equilibrated with buffer D containing 0.15 M NaCl. Sepharose column were pooled and dialyzed against buffer F for 12 The sample was washed with buffer D and eluted with a linear hr and loaded onto a Mono S column. The column was then washed gradient of 0.15-0.8 M NaCl in 30 ml of buffer D. Fractions with 4 ml ofbuffer F, and the sample was eluted with a lineargradient of 0.05-0.5 M NaCI in buffer F and dialyzed against 50%o TGED. 9 and 10 containing the inhibitor (see Fig. 1) were pooled and Aliquots (10 ul) from every other fraction ranging from fractions 24 dialyzed against buffer F (30 mM potassium phosphate, pH to 60 were incubated with 1.5 units of DNA ligase I and 400 ng of 6.8/15% glycerol/10 mM dithiothreitol/50 mM NaCl). The linearized pRSV-neo plasmid and analyzed by agarose gel- sample was applied to a Mono S HR 5/5 column, washed with electrophoresis. Lanes: A, plasmid only; B, plasmid with DNA ligase 4.0 ml of buffer F, and eluted with a linear gradient of I; 24-60, plasmid mixed with DNA ligase I and even-numbered 0.05-0.5 M NaCl in 24 ml of buffer F; 0.3-ml fractions were protein fractions from the Mono S column. collected. Fractions 44-50 containing the inhibitor (see Fig.
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