To gain further insights into the biological functions of Dna2, previously known as a cellular replicative helicase in Saccharomyces cerevisiae, we examined biochemical properties of the recombinant Dna2 protein purified to homogeneity. Besides the single-stranded (ss) DNA-dependent ATPase activity as reported previously, we were able to demonstrate that ssDNA-specific endonuclease activity is intrinsically associated with Dna2. Moreover, Dna2 was capable of degrading duplex DNA in an ATP-dependent fashion. ATP and dATP, the only nucleotides hydrolyzed by Dna2, served to stimulate Dna2 to utilize duplex DNA, indicating their hydrolysis is required. Dna2 was able to unwind short duplex only under the condition where the endonuclease activity was minimized. This finding implies that Dna2 unwinds only partially the 3′-end of duplex DNA and generates a stretch of ssDNA of limited length, which is subsequently cleaved by the ssDNA-specific endonuclease activity. A point mutation at the conserved ATP-binding site of Dna2 inactivated concurrently ssDNA-dependent ATPase, ATP-dependent nuclease, and helicase activities, indicating that they all reside in Dna2 itself. By virtue of its nucleolytic activities, the Dna2 protein may function in the maintenance of chromosomal integrity, such as repair or other related process, rather than in propagation of cellular replication forks. To gain further insights into the biological functions of Dna2, previously known as a cellular replicative helicase in Saccharomyces cerevisiae, we examined biochemical properties of the recombinant Dna2 protein purified to homogeneity. Besides the single-stranded (ss) DNA-dependent ATPase activity as reported previously, we were able to demonstrate that ssDNA-specific endonuclease activity is intrinsically associated with Dna2. Moreover, Dna2 was capable of degrading duplex DNA in an ATP-dependent fashion. ATP and dATP, the only nucleotides hydrolyzed by Dna2, served to stimulate Dna2 to utilize duplex DNA, indicating their hydrolysis is required. Dna2 was able to unwind short duplex only under the condition where the endonuclease activity was minimized. This finding implies that Dna2 unwinds only partially the 3′-end of duplex DNA and generates a stretch of ssDNA of limited length, which is subsequently cleaved by the ssDNA-specific endonuclease activity. A point mutation at the conserved ATP-binding site of Dna2 inactivated concurrently ssDNA-dependent ATPase, ATP-dependent nuclease, and helicase activities, indicating that they all reside in Dna2 itself. By virtue of its nucleolytic activities, the Dna2 protein may function in the maintenance of chromosomal integrity, such as repair or other related process, rather than in propagation of cellular replication forks. single-stranded DNA double-stranded DNA single-stranded circular histidine and Xpress-epitope-tagged recombinant Dna2 protein fast performance liquid chromatography dithiothreitol bovine serum albumin base pair(s) nucleotide polyacrylamide gel electrophoresis polymerase chain reaction. Maintaining the integrity of chromosomal DNA in eukaryotes is of critical importance to the cell and requires a series of complicated enzymatic processes. This is reflected in the complexity and redundancy of the enzyme systems that participate in DNA metabolism, such as replication, repair, and recombination (1Kornberg A. Baker T.A. DNA Replication. 2nd Ed. W. H. Freeman & Co., New York1992Google Scholar, 2Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology, Washington, D. C.1995Google Scholar). In addition, DNA metabolism is tightly linked to cellular control pathways that regulate the cell division cycle (3Murray A. Hunt T. The Cell Cycle: An Introduction. Oxford University Press, New York1993: 135-152Google Scholar, 4Hartwell L.H. Kastan M.B. Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2316) Google Scholar, 5Murray A.W. Curr. Opin. Genet. & Dev. 1995; 5: 5-11Crossref PubMed Scopus (138) Google Scholar, 6Elledge S.J. Science. 1996; 274: 1664-1672Crossref PubMed Scopus (1772) Google Scholar, 7Kaufmann W.K. Paules R.S. FASEB J. 1996; 10: 238-247Crossref PubMed Scopus (255) Google Scholar, 8Lydall D. Weinert T. Curr. Opin. Genet. & Dev. 1996; 6: 4-11Crossref PubMed Scopus (64) Google Scholar, 9Weinert T. Cancer Surv. 1997; 29: 109-132PubMed Google Scholar). One of the enzymes required to achieve DNA replication, repair, or recombination is the DNA helicase, which uses the energy of ATP to translocate in a specific direction along a DNA strand melting the duplex regions it encounters (10Matson S.W. Kaiser-Rogers K.A. Annu. Rev. Biochem. 1990; 59: 289-329Crossref PubMed Scopus (337) Google Scholar, 11Thömmes P. Hübscher U. Chromosoma (Berl.). 1992; 101: 467-473Crossref PubMed Scopus (48) Google Scholar, 12Lohman T.M. Bjornson K.P. Annu. Rev. Biochem. 1996; 65: 169-214Crossref PubMed Scopus (672) Google Scholar, 13Tuteja N. Tuteja R. Nat. Genet. 1996; 13: 11-12Crossref PubMed Scopus (75) Google Scholar, 14West S.C. Cell. 1996; 86: 177-180Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The single-stranded DNA (ssDNA)1generated by the helicase is utilized by other enzymes that participate in the subsequent steps in DNA metabolic pathways. Recently, theDNA2 gene of Saccharomyces cerevisiae was implicated in chromosomal DNA replication (15Budd M.E. Campbell J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7642-7646Crossref PubMed Scopus (151) Google Scholar, 16Budd M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 1995; 270: 26766-26769Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). DNA2 was originally identified by screening for cell division cycle mutants of S. cerevisiae and was shown to be essential for cell viability and to encode a 172-kDa protein with characteristic DNA helicase motifs (15Budd M.E. Campbell J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7642-7646Crossref PubMed Scopus (151) Google Scholar). Analyses of a temperature-sensitive mutant of DNA2 demonstrated that the mutant cell arrested in the S phase of the cell cycle and was deficient in DNA synthesis but not RNA synthesis upon shift to the nonpermissive temperature (15Budd M.E. Campbell J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7642-7646Crossref PubMed Scopus (151) Google Scholar). Immunoaffinity purified Dna2 fusion protein displayed a DNA-dependent ATPase activity as well as 3′ to 5′ DNA helicase activity specific for fork-structured substrates (15Budd M.E. Campbell J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7642-7646Crossref PubMed Scopus (151) Google Scholar). In addition, a mutation in the ATP binding motif of DNA2 led to the inactivation of the ATPase and helicase activities and rendered the mutant cell inviable (16Budd M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 1995; 270: 26766-26769Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). These findings recommend the Dna2 protein as a candidate for a cellular replicative DNA helicase. Therefore, we decided to study Dna2 in the hopes of gaining understanding of initiation events of DNA replication in eukaryotes. Despite the results reported previously, it was not demonstrated unambiguously that Dna2, by itself, constituted a DNA helicase. The enzyme preparation used in these studies was obtained by an immunoaffinity purification step and contained other protein(s). One such protein present in the Dna2 enzyme preparation was yeast Fen1 (yFen1, also called Rad27/Rth1), a structure-specific endonuclease. A specific association of yFen1 and Dna2 was demonstrated both genetically and biochemically (17Budd M.E. Campbell J.L. Mol. Cell. Biol. 1997; 17: 2136-2142Crossref PubMed Scopus (193) Google Scholar). The Fen1 protein is a multifunctional enzyme found in various organisms; it has been shown to be involved in (i) Okazaki fragment maturation in lagging-strand DNA synthesis in human and simian virus 40 replication in vitro(also called 5′- to 3′-exonuclease, DNase IV, or MF1) (18Ishimi Y. Claude A. Bullock P. Hurwitz J. J. Biol. Chem. 1988; 263: 19723-19733Abstract Full Text PDF PubMed Google Scholar, 19Waga S. Bauer G. Stillman B. J. Biol. Chem. 1994; 269: 10923-10934Abstract Full Text PDF PubMed Google Scholar, 20Li X. Li J. Harrington J. Lieber M.R. Burgers P.M. J. Biol. Chem. 1995; 270: 22109-22112Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 21Rumbaugh J.A. Murante R.S. Shi S. Bambara R.A. J. Biol. Chem. 1997; 272: 22591-22599Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), (ii) an alternative pathway for completion of DNA base-excision repair in human cells (22Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (666) Google Scholar), and (iii) maintenance of dinucleotide repeat stability in yeasts (23Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). Given these functions for Fen1, the observation that Dna2 is associated biochemically with yFen1 suggests that Dna2 is not directly involved in advancing replication forks as suggested from previous studies (15Budd M.E. Campbell J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7642-7646Crossref PubMed Scopus (151) Google Scholar, 16Budd M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 1995; 270: 26766-26769Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Rather, it is likely to participate in one of the other key aspects of DNA metabolism. In addition to its true physiological function in vivo, two important issues related to the native structure of Dna2 have not been resolved yet: (i) are there any unidentified polypeptide(s) other than yFen1 that are associated with Dna2, and (ii) if there are any, do the associated protein(s) influence the biochemical activities of Dna2? It is, therefore, necessary to define the biochemical activities of Dna2 protein alone in order to address the issues involved in the native structure of the Dna2 protein. For this purpose, we clonedS. cerevisiae DNA2 using a gap repair strategy and constructed a recombinant baculovirus in order to overexpress Dna2 protein in insect cells. The initial biochemical characterization of recombinant Dna2 purified to homogeneity gave rise to the unexpected finding that it possessed an intrinsic endonuclease activity in addition to the ssDNA-dependent ATPase activity reported previously. Interestingly, Dna2 appeared to melt the duplex DNA only partially, based on the observation that the 3′-end duplex was susceptible to the endonucleolytic activity of Dna2. This finding suggests that the Dna2 protein by itself does not possess marked DNA helicase activity. If it is to participate in DNA replication as a processive DNA helicase, it may require an additional protein(s) such as yFen1 or other unidentified protein(s) that enable Dna2 to attain a vigorous DNA unwinding activity. Alternatively, Dna2 may be involved in other essential aspects of DNA metabolism that require its unique endonuclease activity. All oligonucleotides used for the construction of various DNA substrates (20, 24, 26, 27, 52, 73, and 98 nucleotides (nt) in length) and for PCR primers to clone the DNA2 gene were synthesized commercially (BioServe, MD). Oligonucleotides greater than 30-mer were gel-purified prior to use. The 52- and 73-mers were described elsewhere (24Park J.S. Choi E. Lee S.H. Lee C. Seo Y.S. J. Biol. Chem. 1997; 272: 18910-18919Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The 24-mer (5′-CGG CAA ATG GAA CGC ACA TGC GCA-3′) was complementary to the 5′-end region of the 73-mer, and the 26-mer (5′-GGA AAA CAT TAT TAA TGG CGT CGA GC-3′) was complementary to the 3′-end region of the 73-mer (see Fig. 6 D for the substrate constructed with these oligonucleotides). The 98-mer (5′-GAA TAC AAG CTT GGG CTG CAG GTC GAC TCT AGA GGA TCC CCG GGC GAG CTC GAA TTC CGG TCT CCC TAT AGT GAG TCG TAT TAA TTT CGA TAA GCC AG-3′) contained 5′-end sequences complementary to 20-mer (5′-CTG CAG CCC AAG CTT GTA TT-3′) and 3′-end to 27-mer (5′-CTG GCT TAT CGA AAT TAA TAC GAC TCA-3′). The oligonucleotides used as PCR primers were as follows: Dna2A (5′-CCG GAA TTC GGA ACT ACT TCA AAG CTA C-3′) and Dna2B (5′-CGC GGA TCC TGA CGA TCT CTT CAA TTG-3′) were used to amplify a 5′-flanking region of the DNA2 gene, whereas Dna2C (5′-CGC GGA TCC ATA ACA GGA AAG CTA TCA C-3′) and Dna2D (5′-CTA GTC TAG AGT TGC TTG GTG CCA CGA-3′) were used for a 3′-flanking region of the gene; Dna2E (5′-CGCGGATCC ATG CCC GGA ACG CCA CAG AA-3′) and Dna2F (5′-CCG GAA TTC AGT CGA TTA GGG ACT ATA G-3′) were used to introduce BamHI site (underlined) at the initiation codon (bold type) of the DNA2 gene. ΦX174 sscDNA was purchased from New England Biolabs. Nucleoside triphosphates were obtained from Boehringer Mannheim and [α-32P]dCTP (6000 Ci/mmol) and [γ-32P]ATP (>5000 Ci/mmol) were purchased from Amersham Pharmacia Biotech. The following proteins were obtained commercially: restriction endonucleases, the Klenow fragment of Escherichia coli DNA polymerase I, and terminal deoxynucleotidyltransferase were from Promega, and polynucleotide kinase was from Bio-Rad. DNA substrates used to examine the DNA unwinding and ATP-dependent nuclease activities of the Dna2 protein were prepared by hybridizing the 52-mer or 73-mer to ΦX174 sscDNA as described (24Park J.S. Choi E. Lee S.H. Lee C. Seo Y.S. J. Biol. Chem. 1997; 272: 18910-18919Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). A partial duplex substrate used to quantify the endonuclease activity of Dna2 was prepared by annealing both 20- and 27-mers (40 pmol each) to the 98-mer (10 pmol) under the conditions as described previously (24Park J.S. Choi E. Lee S.H. Lee C. Seo Y.S. J. Biol. Chem. 1997; 272: 18910-18919Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This substrate contained a single-stranded region (49-nt) flanked by duplex regions (20- and 27-base pairs (bp)). The 3′-ends of the two short oligonucleotides annealed to the 98-mer were labeled by incorporating [α-32P]dCTP, chased with excess unlabeled dCTP in the presence of the Klenow fragment. The substrate was gel-purified prior to use, and its specific activity was approximately 3,000 cpm/fmol. Substrates used to analyze the ssDNA-specific endonuclease activity of Dna2 were prepared by annealing either the 24-mer (40 pmol) or 26-mer (40 pmol) to 10 pmol of the 73-mer (named 3′-overhang and 5′-overhang partial duplex, respectively). In order to prepare 3′-overhang partial duplex substrates, the 73-mer (10 pmol) was first labeled at either its 3′-end by incorporating [α-32P]dideoxy-ATP with terminal deoxynucleotidyltransferase or at its 5′-end by incorporating [γ-32P]ATP with polynucleotide kinase. The 73-mers thus labeled were then annealed to the 24-mer (40 pmol). The 5′-overhang partial duplex was labeled with Klenow by incorporating [α-32P]dCTP at the 3′-end of the 73-mer annealed to the 26-mer. In order to construct a substrate with partial duplexes at each end, both the 24- and 26-mers (40 pmol each) were annealed to the 73-mer (10 pmol) and labeled at the 3′-end of the 73-mer as described above for the 5′-overhang partial duplex. These substrates were also gel-purified prior to use. The specific activities of both substrates were comparable and ranged from 2,000 to 3,000 cpm/fmol. Standard assays for measuring DNA-dependent ATPase activity were carried out in a reaction mixture (20 μl) containing 25 mmTris-HCl (pH 7.8), 2 mm MgCl2, 2 mmDTT, 0.25 mg/ml bovine serum albumin (BSA), 250 μm cold ATP, 20 nm [γ-32P]ATP (>5000 Ci/mmol), and 50 ng of M13 sscDNA. After incubation at 37 °C for 10 min, an aliquot (2 μl) was spotted onto a polyethyleneimine-cellulose plate (J. T. Baker, Inc.), which was then developed in 0.5m LiCl, 1.0 m formic acid solution. The products were analyzed using a PhosphorImager (Molecular Dynamics). The reaction mixture (20 μl) used to examine DNA unwinding activity contained 25 mm Tris-HCl (pH 7.8), 5 mmMgCl2, 2 mm DTT, 0.25 mg/ml BSA, 2 mm ATP, and the 3′-32P-labeled partial duplex ΦX174 DNA substrate (15 fmol). After incubation at 37 °C for 10 min, reactions were stopped with 6× stop solution (4 μl; 60 mm EDTA (pH 8.0), 40% (w/v) sucrose, 0.6% SDS, 0.25% bromphenol blue, and 0.25% xylene cyanol). The reaction products were subjected to electrophoresis for 1.5 h at 150 V through 10% polyacrylamide gel containing 0.1% SDS in 0.5× TBE (45 mmTris-base, 45 mm boric acid, 1 mm EDTA). The gel was dried on a DEAE-cellulose paper and subjected to autoradiography. Labeled DNA products were quantitated with the use of a PhosphorImager. The reaction conditions used to examine Dna2 nuclease activity were the same as those for the DNA unwinding reaction except that ATP was omitted. The three substrates used to examine the ssDNA-specific endonuclease activity of Dna2 were the following linear partial duplex DNA substrates: 3′-overhang and 5′-overhang partial duplexes and partial duplex at both ends with internal ssDNA (refer to Fig. 6 for the structures). In order to examine the effects of ATP on the nuclease activity of Dna2, ATP (2 mm) was added to the reaction mixture that contained the 3′-32P-labeled partial duplex ΦX174 DNA (15 fmol). When necessary, the nucleolytic products were subjected to electrophoresis for 1.5 h at 35 watts in 1× TBE through 20% denaturing polyacrylamide gel containing 7 murea and analyzed as described above. The 5′-flanking (397 bp) and 3′-flanking (365 bp) regions of the DNA2 gene were first cloned by PCR with the primers described above and inserted into the yeast plasmid pRS316 (25Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to obtain pDNA2FL. This plasmid was introduced into S. cerevisiae strain YPH499 (MATa, ade2–101, ura3–52, lys2–801, trp1-Δ63, his3-Δ200, leu2-Δ1), and theDNA2 gene was cloned with the use of a gap repair strategy as described (26Muhlrad D. Hunter R. Parker R. Yeast. 1992; 8: 79-82Crossref PubMed Scopus (416) Google Scholar). The retrieved plasmid (pDNA2) contained an intactDNA2 gene, which was confirmed by both restriction and DNA sequencing analyses (27Sanger F. Coulson A.R. Friedmann T. Air G.M. Barrell B.G. Brown N.L. Fiddes J.C. Hutchison C.A. Slocombe P.M. Smith M. J. Mol. Biol. 1978; 125: 225-246Crossref PubMed Scopus (467) Google Scholar) of the 5′- and 3′-regions. A BamHI site was introduced by using a PCR-amplified fragment that contained aBamHI site prior to the start codon (see above for the sequence of PCR primer Dna2E), and then the DNA2 gene was subcloned into the BamHI site of pBlueBacHis2A (Invitrogen) to generate pHX-DNA2. To prepare DNA2 with N-terminal 405 amino acid deletion, the NdeI-EcoRI fragment of pHX-DNA2 was subcloned into pBlueBacHis2C (Invitrogen) cleaved withBamHI and EcoRI. The ssDNA overhangs generated byNdeI and BamHI were made blunt using Klenow prior to the second cleavage with EcoRI. Recombinant baculoviruses were constructed as recommended by the manufacturer (Invitrogen). The recombinant baculoviruses encoded wild type DNA2 and mutantDNA2 with N-terminal 405-amino acid deletion produce recombinant wild type and mutant Dna2 proteins (HX-Dna2 and HX-Dna2Δ405N, respectively) with additional 37 amino acid residues (MPRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGS). These included six histidines (bold type) and the Xpress epitope (underlined) fused to its N-terminal methionine to facilitate detection and purification of the recombinant Dna2 proteins. The recombinant baculoviruses containing theDNA2 gene were infected into Hi-5 insect cells for 48 h at a multiplicity of infection of 10. The infected cells (1 × 106 cells/ml, 2 liters) were harvested, resuspended in 160 ml of buffer T (25 mm Tris-HCl (pH 7.5), 1 mmEDTA, 10% glycerol, 1 mm DTT, 0.1 mm PMSF, 0.15 μg/ml leupeptin and antipain) containing 100 mmNaCl, and disrupted by sonication (7 cycles of a 30-s pulse and a 2-min cooling interval). The extracts (3.35 mg/ml) were cleared by centrifuging at 37,000 rpm for 1 h in a Beckman 45 Ti rotor, and the supernatant was applied directly to a heparin-Sepharose (Amersham Pharmacia Biotech) column (2.5 × 8 cm, 39.2 ml) equilibrated with buffer T plus 100 mm NaCl (buffer T100, hereafter, the number indicates the concentration of NaCl added to buffer T). The column was washed with 10 volumes of the same buffer containing neither DTT nor EDTA and eluted with buffer T600 (−DTT, −EDTA) plus 5 mm imidazole. The peak protein (1.82 mg/ml, 45 ml) was pooled and loaded onto a Ni2+-NTA agarose (Quiagen) column (1.5 × 3.5 cm, 6.2 ml) equilibrated with buffer T600 (−DTT, −EDTA) plus 5 mm imidazole. After extensive washing with buffer T600 (−DTT, −EDTA) plus 20 mm imidazole, the column was eluted with a 60-ml linear gradient of 20–400 mm imidazole in buffer T600 (−DTT, −EDTA). Fractions containing the DNA-dependent ATPase activity were pooled and dialyzed for 3 h against buffer T100. The dialysate (0.25 mg/ml, 180 ml) was loaded onto an SP-Sepharose column (Amersham Pharmacia Biotech) (1.5 × 3 cm, 5.3 ml) equilibrated with buffer T100. After washing with 5 column volumes of buffer T100, the column was eluted with a 50-ml linear gradient of 100–600 mm NaCl in buffer T. The DNA-dependent ATPase activity, which peaked at 300 mm NaCl, was pooled (1.0 mg/ml, 20 ml) and concentrated 5-fold. Aliquots (250-μl) were applied to glycerol gradients (5 ml, 15–35% glycerol in buffer T) in the presence of 500 mm NaCl (high salt gradient) and subjected to centrifugation for 24 h at 45,000 rpm in a Beckman SW55 Ti rotor. Fractions (220 μl) were collected from the bottom of the gradients and assayed for the DNA-dependent ATPase activity and nuclease activities. The active fractions, which contained more than 90% of the total DNA-dependent ATPase activity, were pooled and stored at −80 °C. One microgram of the purified enzyme hydrolyzed 8.9 nmol of ATP/min at 37 °C, and this preparation was used to examine the ATPase and nuclease activities, unless otherwise stated. A 5′-32P label at the substrate duplex end was not removed after prolonged incubation with this preparation, thus the pool did not contain phosphatase activity. HX-Dna2Δ405N was purified employing the same procedure used for HX-Dna2 as above. In order to confirm that the endonuclease and ATP-dependent nuclease activities copurify with HX-Dna2, additional purification steps were introduced. Active fractions (1.0 ml, 700 μg/ml) obtained from high salt glycerol gradients were adjusted to 100 mmNaCl with buffer T and directly loaded onto FPLC Mono Q (1 ml, Amersham Pharmacia Biotech) column. The column was washed and then eluted with a 20-ml linear gradient of 100–600 mm NaCl in buffer T. The peak fractions (1.0 ml, 145 μg/ml) were concentrated 5-fold and loaded to a second glycerol gradient (5 ml, 15 to 35% glycerol in buffer T) in the absence of NaCl (low salt gradient). The centrifugation was performed, and the resulting fractions were analyzed as described for the high salt gradient. A monoclonal antibody against the Xpress epitope was purchased from Invitrogen. Polyclonal antibody specific for Dna2 was generated as follows: HX-Dna2 (2 mg) from the high salt gradient was subjected to 8% SDS-PAGE, and proteins were visualized by Coomassie staining. Gel slices containing protein were pulverized, then emulsified in (MPL + TDM + CWS) adjuvant (Sigma), and injected into two rabbits (3.5 kg, New Zealand White) according to the manufacturer's instructions (250 μg, each injection). At 21 and 50 days after the primary injections, rabbits were injected a second time (a "boost") with equal amounts of HX-Dna2. Sera were collected 7 days after boosting. The IgG fraction was purified with the use of protein-A Sepharose (Amersham Pharmacia Biotech) as described (28Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 309-311Google Scholar). Crude extracts prepared from insect cells infected with the recombinant Dna2 baculovirus contained significant levels (approximately 1to 3% of total proteins) of full-length HX-Dna2 protein (Fig. 1, lane 2), whereas HX-Dna2 was not observed in control extracts prepared from uninfected cells (Fig. 1, lane 1). The expression of recombinant HX-Dna2 was confirmed by Western blot analysis using two antibodies, anti-Dna2 polyclonal antibody (anti-Dna2) (Fig. 1, lane 5), which was raised against full-length HX-Dna2, and anti-Xpress monoclonal antibody (anti-Xpress) (Fig. 1, lane 9), which only detects protein with an intact N terminus. Uninfected cell extracts did not contain any material that cross-reacted with either of the two antibodies (Fig. 1,lanes 4 and 8), whereas purified protein was detected by both antibodies (Fig. 1, lanes 6 and 7, anti-Dna2; lane 10, anti-Xpress). A collection of polypeptides was present in the crude extracts (Fig. 1, lanes 5 and 9), as well as in the purified enzyme preparation obtained from the high salt gradient (Fig. 1, lane 6). These polypeptides apparently arose by proteolysis, as they cross-reacted with anti-DNA2 or anti-Xpress. Our control antibody (polyclonal antibody directed against bovine papillomavirus (E1) did not cross-react with HX-Dna2 at all (data not shown). Because we observed elevated ssDNA-dependent ATPase activity in the baculovirus-infected cell extracts, we decided to purify HX-Dna2 from crude extracts by monitoring ATP hydrolysis in the presence and absence of ssDNA. The protocol for isolation of HX-Dna2 from crude extracts is as summarized under "Experimental Procedures." The initial purification steps included heparin-Sepharose, a Ni2+-NTA agarose column, SP-Sepharose, and glycerol gradient sedimentation in the presence of 0.5m NaCl (high salt gradient). Fractions eluted from the Ni2+-NTA agarose column that were enriched for a 172-kDa polypeptide also showed an increase in the specific activity of ATPase (data not shown). The high salt gradient did not display a further increase the specific activity of HX-Dna2 (data not shown), indicating that the enzyme preparation was maximally pure at this stage of purification. High salt gradient fractions containing the ATPase activity coincided with those that carried the 172-kDa protein (data not shown). An SDS-PAGE analysis of Mono Q column fractions yielded results identical to those above in that the 172-kDa polypeptide (Fig. 2 A) copurified with the ssDNA-dependent ATPase activity (data not shown). As shown in Fig. 2, we investigated whether HX-Dna2 was able to displace the 73-base oligonucleotide annealed to ΦX174 sscDNA (5′-tailed substrate with a non-complementary 21-nt tail at the 5′-end). When the DNA unwinding activity of HX-Dna2 was examined in the presence (Fig. 2 B) and absence of ATP (Fig. 2 C), we obtained a somewhat puzzling observation. The DNA species generated by HX-Dna2 were not dependent on the presence of ATP (Fig. 2, B and C) but required MgCl2 in the reaction mixture (data not shown). In addition, the DNA products did not appear to correspond to the 73-base oligonucleotide, as they were not uniform in their migration; rather, migration retardation of these DNA was inversely proportional to the amount of HX-Dna2 present in each fraction (Fig. 2, A and B). Notable was the observation that the 32P label at the 3′-end of the annealed oligonucleotide was released in an ATP-dependent fashion when HX-Dna2 concentration was enriched (compare Fig. 2,B and C). This observation suggests that the enzyme may possess an exonucleolytic activity that is able to act on dsDNA in the 3′ to 5′ direction and in an ATP-dependent manner. This activity, termed ATP-dependent nuclease activity, of HX-Dna2 was analyzed further and is discussed in detail below. Taken together, our observation suggests that the DNA species observed in Fig. 2 most likely result from a nuclease activity of HX-Dna2, indicating that Dna2 possesses at least one type of nuclease activity but not a strong DNA unwinding activity. However, we were able to detect a weak DNA unwinding activity using a 20-mer oligonucleotide-annealed ΦX174 sscDNA as substrate under the condition where the endonuclease activity was inhibited (see below; Fig. 9)Figure 9DNA unwinding activity of HX-Dna2 protein is observed in a condition where nuclease activity is suppressed.Schematic structure of the partial duplex substrate (20-mer (5′-GGC GAT TGC GTA CCC GAC GA-3′) annealed to ΦX174 sscDNA) is shown atleft. The asterisk indicates a radioisotopic label at the 3′-end. HX-Dna2K1080E has a substitution of glutamate for the conserved lysine residue in the ATP binding motif as reported (16Budd M.E. Choe W.C. Campbell J.L. J. Biol. Chem. 1995; 270: 26766-26769Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). HX-Dna2Δ405N has a deletion of N-terminal 405 amino acids as described under "Experimental Procedures." Enzymes (40 ng of HX-Dna2 and HX-Dna2K1080E; 20 ng of HX-Dna2 Δ405N) were added to reaction mixtures (20 μl) containing 15 fmol of the 20-mer annealed ΦX174 substrate in the presence of indicated amounts of Mg2+ and ATP and incubated at 37 °C for 10 min. After incubation, the products were subjected to electrophoresis for 1.5 h at 150 V through 12% polyacrylamide gel containing 0.1% SDS in 0.5× TBE. S and B denote substrate alone and boiled substrate controls, respectively. The arrow indicates the position where the 20-mer oligonucleotide migrated. The amounts of substrate unwound and 3′-end labels released were measured with the use of a PhosphorImager, and the results are presented at thebottom of the figure.View Large Image Figure ViewerDownload Hi-res image
AIMTo evaluate a novel grading system to predict lymph node metastasis (LNM) in patients with submucosal invasive colorectal carcinoma (SICRC). METHODSWe analyzed the associations between LNM and various clinicopathological features in 252 patients with SICRC who had undergone radical surgery at the Seoul Saint Mary's hospital between 2000 and 2015. RESULTSLNM was observed in 31 patients (12.3%).The depth and width of the submucosal invasion, lymphatic invasion, tumor budding, and the presence of poorly differentiated clusters (PDCs) were significantly 5936August 28, 2017|Volume 23|Issue 32| WJG|www.wjgnet.com
<p>Supplementary Fig. S5. Validation of redundant APC mutations. A, For one case (CRC2), two redundant APC mutations are validated by Sanger sequencing. One nonsense mutation (C>T; left) and one frameshift indel (AG>A; right) are shown for seven regional biopsies for the given case. For one primary case (CRC2-P1), the nonsense mutation is not observed, consistent with the metastasis-clonal presentation of this mutation. B, The copy number changes of the entire chromosome 5 are shown for seven regional biopsies. Entire loss of chromosome 5 is observed for CRC-P1. APC locus is incidated with a red dotted line.</p>
<p>Supplementary Fig. S4. Immunohistochemical identification of decreased PCDH9 expression in liver metastasis of a colon cancer. A colon cancer with ITH of PCDH9 deletion identified by both aCGH and qPCR shows positive PCDH9 immunostaining in normal colonic glands (A) and a primary colon tissue (B), but decreased PCDH9 immunostaining in two metastatic sites (C and D) in the liver (frozen tissues).</p>
<div>AbstractPurpose:<p>Histologic features of diffuse-type gastric cancer indicate that the tumor microenvironment (TME) may substantially impact tumor invasiveness. However, cellular components and molecular features associated with cancer invasiveness in the TME of diffuse-type gastric cancers are poorly understood.</p>Experimental Design:<p>We performed single-cell RNA-sequencing (scRNA-seq) using tissue samples from superficial and deep invasive layers of cancerous and paired normal tissues freshly harvested from five patients with diffuse-type gastric cancer. The scRNA-seq results were validated by immunohistochemistry (IHC) and duplex <i>in situ</i> hybridization (ISH) in formalin-fixed paraffin-embedded tissues.</p>Results:<p>Seven major cell types were identified. Fibroblasts, endothelial cells, and myeloid cells were categorized as being enriched in the deep layers. Cell type–specific clustering further revealed that the superficial-to-deep layer transition is associated with enrichment in inflammatory endothelial cells and fibroblasts with upregulated <i>CCL2</i> transcripts. IHC and duplex ISH revealed the distribution of the major cell types and CCL2-expressing endothelial cells and fibroblasts, indicating tumor invasion. Elevation of CCL2 levels along the superficial-to-deep layer axis revealed the immunosuppressive immune cell subtypes that may contribute to tumor cell aggressiveness in the deep invasive layers of diffuse-type gastric cancer. The analyses of public datasets revealed the high-level coexpression of stromal cell–specific genes and that <i>CCL2</i> correlated with poor survival outcomes in patients with gastric cancer.</p>Conclusions:<p>This study reveals the spatial reprogramming of the TME that may underlie invasive tumor potential in diffuse-type gastric cancer. This TME profiling across tumor layers suggests new targets, such as CCL2, that can modify the TME to inhibit tumor progression in diffuse-type gastric cancer.</p><p><i>See related commentary by Huang and Brekken, p. 6284</i></p></div>
// Seung-Hyun Jung 1, 3 , Min Sung Kim 2 , Chan Kwon Jung 4 , Hyun-Chun Park 1, 3 , So Youn Kim 1, 3 , Jieying Liu 1, 3 , Ja-Seong Bae 5 , Sung Hak Lee 4 , Tae-Min Kim 6 , Sug Hyung Lee 2 , Yeun-Jun Chung 1, 3 1 Department of Microbiology, College of Medicine, The Catholic University of Korea, Seoul, Korea 2 Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea 3 Department of Integrated Research Center for Genome Polymorphism, College of Medicine, The Catholic University of Korea, Seoul, Korea 4 Department of Hospital Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea 5 Department of General Surgery, College of Medicine, The Catholic University of Korea, Seoul, Korea 6 Department of Medical Informatics, College of Medicine, The Catholic University of Korea, Seoul, Korea Correspondence to: Yeun-Jun Chung, email: yejun@catholic.ac.kr Sug Hyung Lee, email: suhulee@catholic.ac.kr Keywords: follicular thyroid adenoma, follicular thyroid carcinoma, mutations, copy number alteration, tumor progression Received: May 20, 2016 Accepted: September 02, 2016 Published: September 09, 2016 ABSTRACT Follicular thyroid adenoma (FTA) precedes follicular thyroid carcinoma (FTC) by definition with a favorable prognosis compared to FTC. However, the genetic mechanism of FTA to FTC progression remains unknown. For this, it is required to disclose FTA and FTC genomes in mutational and evolutionary perspectives. We performed whole-exome sequencing and copy number profiling of 14 FTAs and 13 FTCs, which exhibited previously-known gene mutations ( NRAS, HRAS, BRAF, TSHR and EIF1AX ) and copy number alterations (CNAs) (22q loss and 1q gain) in follicular tumors. In addition, we found eleven potential cancer-related genes with mutations ( EZH1, SPOP, NF1, TCF12, IGF2BP3, KMT2C, CNOT1, BRIP1, KDM5C, STAG2 and MAP4K3 ) that have not been reported in thyroid follicular tumors. Of note, FTA genomes showed comparable levels of mutations to FTC in terms of the number, sequence composition and functional consequences (potential driver mutations) of mutations. Analyses of evolutionary ages using somatic mutations as molecular clocks further identified that FTA genomes were as old as FTC genomes. Whole-transcriptome sequencing did not find any gene fusions with potential significance. Our data indicate that FTA genomes may be as old as FTC genomes, thus suggesting that follicular thyroid tumor genomes during the transition from FTA to FTC may stand stable at genomic levels in contrast to the discernable changes at pathologic and clinical levels. Also, the data suggest a possibility that the mutational profiles obtained from early biopsies may be useful for the molecular diagnosis and therapeutics of follicular tumor patients.
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