FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1978620556> ?p ?o ?g. }

• W1978620556 endingPage "25214" @default.
• W1978620556 startingPage "25205" @default.
• W1978620556 abstract "β protein from bacteriophage λ promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. β protein binds most tightly to an intermediate of annealing formed by the sequential addition of two complementary oligonucleotides. Here we have characterized the domain structure of β protein in the presence and absence of DNA using limited proteolysis. Residues 1–130 form an N-terminal “core” domain that is resistant to proteases in the absence of DNA, residues 131–177 form a central region with enhanced resistance to proteases upon DNA complex formation, and the C-terminal residues 178–261 of β protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of β protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of β protein are protected from biotinylation in the DNA complex, whereas none of the lysine residues in the C-terminal portion are protected. The results lead to a model for the domain structure and DNA binding of β protein in which a stable N-terminal core and a more flexible central domain come together to bind DNA, whereas a C-terminal tail remains disordered. A fragment consisting of residues 1–177 of β protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA. β protein from bacteriophage λ promotes a single-strand annealing reaction that is central to Red-mediated recombination at double-strand DNA breaks and chromosomal ends. β protein binds most tightly to an intermediate of annealing formed by the sequential addition of two complementary oligonucleotides. Here we have characterized the domain structure of β protein in the presence and absence of DNA using limited proteolysis. Residues 1–130 form an N-terminal “core” domain that is resistant to proteases in the absence of DNA, residues 131–177 form a central region with enhanced resistance to proteases upon DNA complex formation, and the C-terminal residues 178–261 of β protein are sensitive to proteases in both the presence and absence of DNA. We probed the DNA binding regions of β protein further using biotinylation of lysine residues and mass spectrometry. Several lysine residues within the first 177 residues of β protein are protected from biotinylation in the DNA complex, whereas none of the lysine residues in the C-terminal portion are protected. The results lead to a model for the domain structure and DNA binding of β protein in which a stable N-terminal core and a more flexible central domain come together to bind DNA, whereas a C-terminal tail remains disordered. A fragment consisting of residues 1–177 of β protein maintains normal binding to sequentially added complementary oligonucleotides and has significantly enhanced binding to single-strand DNA. The Red system of bacteriophage λ consists of three proteins that promote DNA recombination initiated at dsDNA 4The abbreviations used are: dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; DTT, dithiothreitol; HPLC, high performance liquid chromatography; LC, liquid chromatography; MS, mass spectroscopy.4The abbreviations used are: dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; DTT, dithiothreitol; HPLC, high performance liquid chromatography; LC, liquid chromatography; MS, mass spectroscopy. breaks or at the overlapping ends of the linear λ chromosome (1Poteete A.R. FEMS Microbiol. Lett. 2001; 201: 9-14PubMed Google Scholar, 2Kuzminov A. Annu. Rev. Genet. 1999; 28: 49-70Google Scholar). The exo gene (redα) encodes λ exonuclease, a 24-kDa protein with 5′-3′ exonuclease activity (3Little J.W. J. Biol. Chem. 1967; 242: 679-686Abstract Full Text PDF PubMed Google Scholar, 4Carter D.M. Radding C.M. J. Biol. Chem. 1971; 246: 2502-2512Abstract Full Text PDF PubMed Google Scholar). The bet gene (redβ) encodes the 29-kDa β protein, which binds ssDNA and promotes annealing of complementary strands (5Kmiec E. Holloman W.K. J. Biol. Chem. 1981; 256: 12636-12639Abstract Full Text PDF PubMed Google Scholar, 6Muniyappa K. Radding C.M. J. Biol. Chem. 1986; 261: 7472-7478Abstract Full Text PDF PubMed Google Scholar). The gam gene encodes the 16-kDa γ protein, which binds and inhibits host nuclease enzymes (7Marsic N. Roje S. Stojiljkovic I. Salaj-Smic E. Trgovcevic Z. J. Bacteriol. 1993; 175: 4738-4743Crossref PubMed Google Scholar, 8Kulkarni S.K. Stahl F.W. Genetics. 1989; 123: 249-253Crossref PubMed Google Scholar). Together, the three proteins promote recombination events that enhance the replication and repair of phage λ DNA within the Escherichia coli host. Certain strains of E. coli contain an analogous recombination system, RecET, encoded on a cryptic rac prophage, that is turned on to promote recombination in certain genetic backgrounds (9Kolodner R. Hall S.D. Luisi-Deluca C. Mol. Microbiol. 1994; 11: 23-30Crossref PubMed Scopus (76) Google Scholar). RecE and RecT are functionally analogous and related in sequence to λ exonuclease and β protein, respectively (10Iyer L.M. Koonin E.V. Aravind L. BMC Genomics. 2002; 3: 8Crossref PubMed Scopus (140) Google Scholar, 11Aravind L. Makarova K.S. Koonin E.V. Nucleic Acids Res. 2000; 28: 3417-3432Crossref PubMed Google Scholar). Both the RecET and Redαβ systems have in recent years been employed in novel and powerful methods for genetic engineering (12Copeland N.G. Jenkins N.A. Court D.L. Nat. Rev. Genet. 2001; 2: 769-779Crossref PubMed Scopus (596) Google Scholar, 13Muyrers J.P.P. Zhang Y. Stewart A.F. Trends Biochem. Sci. 2001; 26: 325-331Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) and, thus, are of interest technologically in addition to their long-standing as model systems for understanding general principles of genetic recombination.Purified β protein promotes renaturation of complementary strands of DNA (5Kmiec E. Holloman W.K. J. Biol. Chem. 1981; 256: 12636-12639Abstract Full Text PDF PubMed Google Scholar, 6Muniyappa K. Radding C.M. J. Biol. Chem. 1986; 261: 7472-7478Abstract Full Text PDF PubMed Google Scholar) and annealing of shorter oligonucleotides (14Karakousis G. Ye N. Li Z. Chiu S.K. Reddy G. Radding C.M. J. Mol. Biol. 1998; 276: 721-731Crossref PubMed Scopus (67) Google Scholar). The protein binds weakly to ssDNA of minimal length of about 36 nucleotides with a site size of approximately one monomer per five nucleotides (15Mythili E. Kumar K.A. Muniyappa K. Gene. 1996; 182: 81-87Crossref PubMed Scopus (39) Google Scholar). β protein does not bind directly to dsDNA but remains tightly associated with the duplex product of annealing formed by the sequential addition of two complementary oligonucleotides to β protein (14Karakousis G. Ye N. Li Z. Chiu S.K. Reddy G. Radding C.M. J. Mol. Biol. 1998; 276: 721-731Crossref PubMed Scopus (67) Google Scholar). Although β protein is not an ATPase, it will promote some RecA-like strand exchange and invasion reactions in vitro but only when certain restrictive criteria are met, such as high A/T composition (16Li Z. Karakousis G. Chiu S.K. Reddy G. Radding C.M. J. Mol. Biol. 1998; 276: 733-744Crossref PubMed Scopus (78) Google Scholar, 17Rybalchenko N. Golub E.I. Baoyuan B. Radding C.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17056-17060Crossref PubMed Scopus (40) Google Scholar).The structure of β protein has been studied by electron microscopy (18Passy S.I. Yu X. Li Z. Radding C.M. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4279-4284Crossref PubMed Scopus (82) Google Scholar). In the presence of Mg2+, the protein alone forms rings of ∼12 subunits, slightly larger rings (15–18 subunits) in the presence of ssDNA, and left-handed helical filaments in the presence of dsDNA or two complementary strands of ssDNA. A model was proposed in which ssDNA binds to the outer surface of the rings formed by β protein in such a way that the bases are exposed and available for interactions with a second strand of ssDNA, which may be bound by a separate ring of β protein. Initiation of annealing likely nucleates formation of a β protein filament, which coats the duplex product of annealing. Presumably the strongly favorable interaction of β protein with the newly formed dsDNA drives the reaction in favor of annealing.β belongs to a family of proteins (10Iyer L.M. Koonin E.V. Aravind L. BMC Genomics. 2002; 3: 8Crossref PubMed Scopus (140) Google Scholar) including RecT, for which there is no representative crystal structure. β protein and RecT, the best-studied members of this family, share only about 15% sequence identity with one another. Secondary structure prediction based on a multiple sequence alignment suggests four α-helices and five β-strands for a conserved region corresponding to residues 19–205 of β protein (10Iyer L.M. Koonin E.V. Aravind L. BMC Genomics. 2002; 3: 8Crossref PubMed Scopus (140) Google Scholar). Notable features in the sequences include several conserved aromatic and apolar residues and two consecutive highly conserved acidic residues near the end of the conserved region. Beyond the low resolution electron microscopy studies and what can be gleaned from sequence analyses, very little is known about the structure of β protein.Here we have used limited proteolysis, N-terminal sequencing, and mass spectrometry to probe the domain structure of β protein in the presence and absence of DNA. The results reveal a protease-resistant N-terminal core domain, a central region that is stabilized by DNA complex formation, and a flexible C-terminal tail. In addition, application of a recently developed technique for mapping out protein-DNA interactions, which involves biotinylation of lysine residues and mass spectrometry (19Shell S.M. Hess S. Kvaratskhelia M. Zou Y. Biochemistry. 2005; 44: 971-978Crossref PubMed Scopus (40) Google Scholar, 20Kvaratskhelia M. Miller J.T. Budihas S.R. Pannell L.K Le Grice S.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15988-15993Crossref PubMed Scopus (56) Google Scholar), suggests that both the N-terminal and central regions of β protein, but not the C-terminal tail, are involved in binding to DNA.EXPERIMENTAL PROCEDURESExpression and Purification of β Protein—The bet gene encoding β protein was PCR-amplified from phage λ genomic DNA (New England Biolabs) and cloned into pET-14b (Novagen) between the NdeI and BamH1 sites. The primers for PCR amplification were 5′-G GAA TTC CAT ATG AGT ACT GCA CTC GC-3′ (forward) and 5′-CGC GGA TCC TCA TGC TGC CAC CTT CTG C-3′ (reverse). The resulting plasmid expresses β protein with an N-terminal His6 tag followed by a thrombin cleavage site. The integrity of the insert was verified by DNA sequencing, and the plasmid was transformed into BL21 (DE3) pLysS E. coli cells. Cell cultures (6 × 1 liter) were grown in Luria broth at 37 °C to an absorbance at 600 nm of 0.5, induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside, and shaken at 18 °C for an additional 16 h. Cells were harvested by centrifugation at 10,000 × g, and the pellets were stored at –80 °C. Cell pellets were thawed and re-suspended in 50 ml of buffer A (300 mm NaCl, 30 mm NaH2PO4, 10 mm imidazole, pH 8.0) supplemented with 1 mg/ml lysozyme, 100 μg/ml α-phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, and 1 μg/ml leupeptin. After sonication the lysate was centrifuged at 39,000 × g, and the supernatant was loaded onto a 10-ml column of Ni2+-loaded chelating Sepharose fast flow (Amersham Biosciences). The column was washed with 300 ml of buffer A containing 30 mm imidazole, and the protein was eluted with a 200-ml linear gradient from 30–500 mm imidazole in buffer A. Pooled fractions containing β protein were dialyzed into thrombin cleavage buffer (20 mm NaH2PO4, 150 mm NaCl, pH 7.4) and incubated with 50 units of thrombin (Amersham Biosciences) at 22 °C for ∼24 h. The protein was then loaded onto the Ni2+ column and the flow-through was dialyzed into 20 mm Tris, pH 8.0, loaded onto a 25-ml HiTrap Q HP anion exchange column (Amersham Biosciences), and eluted with a 180-ml linear gradient from 0 to 1 m NaCl. Pooled fractions were dialyzed into 20 mm Tris, pH 8.0, 1 mm DTT, concentrated to 47 mg/ml, and stored at –80 °C in small aliquots. The concentration of β protein was determined by absorbance at 280 nm in 6 m guanidine hydrochloride, 0.02 m NaH2PO4, pH 6.5, using an extinction coefficient of 34,850 m–1 cm–1, which was calculated from the amino acid sequence (21Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5022) Google Scholar). The final protein contains the extra N-terminal sequence Gly-Ser-His. The K253A mutant of β protein was generating using the QuikChange procedure (Stratagene), and the protein was purified as described above for wild type.Expression and Purification of β Protein Fragments—Regions of DNA encoding amino acids 1–177 and 1–188 of β protein were PCR-amplified from phage λ genomic DNA and cloned into pET-9a between the NdeI and BamH1 sites. PCR used the forward primer listed above and the reverse primers 5′-CGC GGA TCC TCA ACG TTC TGC AGT GTA TGC-3′ (β1–188) and CGC GGA TCC TCA GCG CTC GGC TTC ATC CTT GTC (β1–177). The K11A, K36A, K69A, and K172A mutants of the β1–188 fragment were generated by the QuikChange procedure. Plasmids were transformed into BL21-AI E. coli cells (Invitrogen), grown in 1-liter cultures of Luria broth at 37 °C to an absorbance at 600 nm of 0.5, induced with 0.2% arabinose, and shaken at 15 °C for an additional 16 h. Cells were harvested by centrifugation, and the pellets were stored at –80 °C.For purification of β1–188 protein, cells were thawed and resuspended in 35 ml of buffer B (500 mm NaCl, 50 mm NaH2PO4, 10% glycerol, 10 mm imidazole, pH 8.0) supplemented with lysozyme and protease inhibitors as described above. After sonication, the lysate was centrifuged at 39,000 × g, and the supernatant was dialyzed into 20 mm Tris, pH 8.0, loaded onto a 25-ml HiTrap Q HP anion exchange column, and eluted with a 180-ml linear gradient from 0 to 1 m NaCl. Pooled fractions were dialyzed into 20 mm NaH2PO4, pH 7.0, loaded onto a 25-ml HiTrap heparin column (Amersham Biosciences), and eluted with a 180-ml linear gradient from 0 to 1.5 m NaCl. Pooled fractions were dialyzed into 20 mm Tris pH 8.0, 1 mm DTT, concentrated to 66 mg/ml, and stored at –80 °C in small aliquots.For purification of β1–177, thawed cells were re-suspended in buffer B. After sonication and centrifugation of the cell lysate, polyethyleneimine was added to the supernatant to a final concentration of 0.5%, and the resulting suspension was stirred at 4 °C for 30 min and centrifuged at 12,000 × g. The supernatant was collected and dialyzed into 20 mm Tris, pH 8.0, 100 mm NaCl. The resulting suspension was centrifuged at 10,000 × g, and the protein from the supernatant was further purified by anion exchange and heparin chromatography as described above for β1–188 except that 100 mm NaCl was included in the buffer before the heparin column. Purification of β1–188 proteins with the K11A, K36A, K69A, and K172A mutations followed this same procedure, except the heparin chromatography step was omitted, and the final buffer included 100 mm NaCl.Oligonucleotides—Two complementary 33-mer oligonucleotides, 33+ (ACA GCA CCA GAT TCA GCA ATT AAG CTC TAA GCC) and 33– (GGC TTA GAG CTT AAT TGC TGA ATC TGG TGC TGT), which are derived from a sequence present in M13 phage DNA (14Karakousis G. Ye N. Li Z. Chiu S.K. Reddy G. Radding C.M. J. Mol. Biol. 1998; 276: 721-731Crossref PubMed Scopus (67) Google Scholar), were purchased from Integrated DNA Technologies and purified by ion exchange HPLC. Oligonucleotide concentrations were determined from absorbance at 260 nm using extinction coefficients calculated from their sequences and are expressed in nucleotides. 33+ oligonucleotide was 32P-5′-end-labeled with T4 polynucleotide kinase (New England Biolabs), and free nucleotides were removed with a MicroSpin G-25 column (Amersham Biosciences).DNA Binding Assays—The gel shift assay to measure binding of β protein to sequentially added oligonucleotides is based on a published protocol (14Karakousis G. Ye N. Li Z. Chiu S.K. Reddy G. Radding C.M. J. Mol. Biol. 1998; 276: 721-731Crossref PubMed Scopus (67) Google Scholar). Each 20-μl reaction contained 50 mm Tris, pH 7.5, 1 mm DTT, and 0.1 mg/ml bovine serum albumin. 32P-end-labeled 33+ and β protein of the indicated concentrations were incubated at 37 °C, and after 10 min the complementary oligonucleotide (33–) of the indicated concentration was added, and the reaction was incubated an additional 30 min at 37 °C. After the addition of 5 μl of 5× loading buffer (20% glycerol, 0.12% bromphenol blue, 0.12% xylene cyanol), each sample was loaded onto a 15% native polyacrylamide gel (5% stacking gel) and electrophoresed in TAE (40 mm Tris acetate, pH 8.3, 1 mm EDTA) buffer at room temperature. Dried gels were autoradiographed.Binding of full-length β protein and the β1–177 fragment to a 33-mer oligonucleotide (33+) was measured using a double-filter method (22Wong I. Lohman T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5428-5432Crossref PubMed Scopus (359) Google Scholar). Binding reactions (50 μl) were in buffer C (20 mm Tris-HCl, pH 7.5, 1 mm MgCl2, 1 mm DTT) and contained ∼100 nm 32P-end-labeled 33+ oligonucleotide and 0–1 mm protein. The reactions were equilibrated at 37 °C for 30 min and then loaded onto a Minifold I 48-well slot-blot apparatus (Schleicher & Schuell) containing nitrocellulose (Bio-Rad) and DEAE (Whatman) filters. Samples were pulled through the filters under vacuum and washed with 1 ml of buffer C. The radioactivity on the filters was measured using a Storm 860 PhosphorImager (Amersham Biosciences) and ImageQuant 5.2 software (Molecular Dynamics). The percentage of protein-bound DNA was calculated from the intensities on the nitrocellulose (protein-bound) and DEAE (unbound) filters.Limited Proteolysis—Limited trypsin digestions were performed in 25 mm Hepes, pH 7.9, 10% glycerol, 0.2 mm EDTA, 5 mm MgCl2, 20 mm CaCl2, and 60 mm KCl at 25 °C. The reactions contained 2.5 mg/ml (86 μm) β protein in the absence or presence of 250 μm each of sequentially added 33+ and 33– oligonucleotides, as described above. Trypsin was added to a final concentration of 5 ng/μl, and 10 μl of the reaction was removed at the indicated times. Reactions were quenched with 10 μl of 2× SDS-PAGE loading buffer, heated at 95 °C for 5 min, electrophoresed on a 13.5% SDS-PAGE gel, and stained with Coomassie Blue. Digestions using the same procedure were also done with 50 ng/μl chymotrypsin, 10 ng/μl subtilisin, and 25 ng/μl thermolysin. For mass spectrometric analysis of limited proteolysis products, reactions were incubated for 30 min, quenched by adding 6 m guanidine hydrochloride, and injected directly onto the LC-MS instrument.For N-terminal sequencing, unstained SDS-PAGE gels were blotted onto polyvinylidene fluoride membranes and subsequently stained with Coomassie Blue. Individual bands were cut out of the gel and sequenced by automated Edman degradation (Protein and Nucleic Acid Chemistry Laboratory, Washington University, St. Louis, MO) using an Applied Biosystems model 494 Procise HT sequencer. Data analysis was performed with Applied Biosystems model 610A software. Standard reagents and conditions were employed.Biotinylation Reactions—12 μl of 2.5 μg/μl β protein or β protein-DNA complex was prepared as described above. 38 μl of phosphate-buffered saline (Sigma) and 2.5 μl of 40 mm NHS-biotin (Pierce) dissolved in Me2SO were mixed and incubated at room temperature for 40 min, and the reactions were quenched by adding 5 μl of 100 mm lysine (8.7 mm final concentration). The biotinylated protein was subjected to complete trypsin digestion as has been described (23Stone K.L. Williams K.R. Walker J.M. The Protein Protocols Handbook. Humana Press Inc., Totowa, NJ2002: 511-521Google Scholar). Briefly, the protein was precipitated from the reaction mixture by adding acetone, and the pellet was washed with acetone and dried. The pellet was dissolved in 20 μl of 8 m urea, 0.4 m NH4HCO3, reduced with 5 μl 45 mm DTT, and alkylated with 5 μl of 100 mm iodoacetic acid. 50 μl of double-distilled H2O was then added to dilute the digestion buffer to 2 m urea, 0.1 m NH4HCO3. 5 μl of 0.2 μg/μl trypsin in 1% acetic acid was added, and the mixture was incubated at 37 °C for 24 h. The trypsin digestion was stopped by adding 10 μl of 2% trifluoroacetic acid. A 10-μl aliquot of the mixture was then subjected to LC-MS analysis.Mass Spectrometry—All mass spectrometric analyses used a ThermoFinnigan LCQ DECA ion trap mass spectrometer coupled with a Surveyor HPLC system (ThermoQuest, San Jose, CA). For mass spectrometry of limited proteolysis reactions, HPLC separations were performed with a PLRP-S column (8 μm, 1000 Å, 1.0 × 150 mm, Michrom Bioresources, Inc.) and a Vydas C4 guard column using H O/acetonitrile gradients at a flow rate of 50 μl/min. 0.1% formic 2acid and 0.02% trifluoroacetic acid were included in both water and acetonitrile. The data were collected in positive ion mode with full scan ranging from 400–2000.For mass spectrometry of trypsin digests of biotinylation reactions, collection of all MS and MS/MS data used a C18 column (Vydas, 1.0-mm inner diameter × 250 mm long, 5-μm particles, and 300 Å pore size; Hesperia, CA) with Vydas C18 guard column. In the survey run the mass spectrometer was operated in data-dependent mode and cycled through a single MS and five MS/MS experiments. MS/MS experiments (collision energy 35%) were performed on the five most abundant ions (3-Da window; precursor m/z ± 1.5 Da) in each full scan mass spectrum. MS/MS spectra were matched to amino acid sequences from β protein using SEQUEST with differential modification of 226.3 Da to lysine and a static modification of 57 Da to cysteine. Once the unbiotinylated and biotinylated peaks were assigned to individual peptides, the retention time and best response charge states were recorded. The mass spectrometer was then switched to full scan ion mode with the scan range of 400–1500. When signal responses of analytes were compared, chromatographic peak area was used. The doubly charged peak of “VGMDSVDPQELITTLR” (16–31)2+ was selected as internal loading control, since this peptide fragment is not affected by biotinylation and is always formed by tryptic digestion. All other peptide responses were normalized to the response of this peptide.RESULTSLimited Proteolysis of β Protein Alone and in Complex with DNA—We first verified that our preparation of β protein, which has the extra N-terminal residues GSH, has the expected DNA binding properties. In the assay (14Karakousis G. Ye N. Li Z. Chiu S.K. Reddy G. Radding C.M. J. Mol. Biol. 1998; 276: 721-731Crossref PubMed Scopus (67) Google Scholar), β protein is first incubated with a 32P-end-labeled 33-mer (33+) oligonucleotide, and as the complementary oligonucleotide (33-) is titrated in, the resulting β protein-DNA complex is shifted to the top of a 15% polyacrylamide gel (supplemental Fig. S1). Binding of β protein to only one oligonucleotide (lane 3) or to pre-annealed duplex 33-mer (not shown) is not observed. Thus, as has been concluded previously (14Karakousis G. Ye N. Li Z. Chiu S.K. Reddy G. Radding C.M. J. Mol. Biol. 1998; 276: 721-731Crossref PubMed Scopus (67) Google Scholar), a tight complex is formed only when the two complementary oligonucleotides are added sequentially to β protein. As seen by electron microscopy, this preparation of β protein also forms rings and filaments in the presence of heat-denatured dsDNA (data not shown) that appear very similar to those reported for native β protein (18Passy S.I. Yu X. Li Z. Radding C.M. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4279-4284Crossref PubMed Scopus (82) Google Scholar).The domain structure of β protein both alone and in complex with DNA was investigated using limited proteolysis with trypsin. In the absence of DNA, β protein is rapidly digested into a distinct fragment that appears as a ∼15-kDa band on the SDS-PAGE gel (Fig. 1). N-terminal sequencing and mass spectrometric analysis of this cleavage product unambiguously identified it to be residues 1–134 of β protein. In complex with annealed duplex DNA (as prepared in Fig. S1, panel A, lane 8), parts of β protein are significantly protected from trypsin digestion. In particular, three new distinct cleavage products are observed as ∼19-, 21-, and 27-kDa bands, which correspond to residues 16–177, 1–177, and 1–230 of β protein, respectively. The fragment of residues 1–177 is the most stable and persists for greater than 2 h.In addition to stabilizing larger fragments of β protein, DNA complex formation also stabilizes the N-terminal domain (residues 1–134) since it persists for 120 min in the presence of DNA but is completely digested within 120 min in the absence of DNA. DNA does not stabilize full-length β protein, since the upper band disappears at the same rate in both the presence and absence of DNA. Mass spectrometric analysis reveals that the upper band from the samples digested in the presence of DNA does not actually correspond to full-length β protein but rather to residues 1–253 that result from removal of residues 254–261 by trypsin cleavage at Lys-253.The single-strand annealing activity and oligomeric properties of β protein have previously been shown to be sensitive to Mg2+ ions (5Kmiec E. Holloman W.K. J. Biol. Chem. 1981; 256: 12636-12639Abstract Full Text PDF PubMed Google Scholar, 15Mythili E. Kumar K.A. Muniyappa K. Gene. 1996; 182: 81-87Crossref PubMed Scopus (39) Google Scholar, 18Passy S.I. Yu X. Li Z. Radding C.M. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4279-4284Crossref PubMed Scopus (82) Google Scholar). However, the limited trypsin digestion patterns in both the presence and absence of DNA are not affected at all by the omission of Mg2+ from the reaction (data not shown). In addition, essentially the same results seen in Fig. 1 are observed when the buffer is the same as that used for the gel-shift assay of Fig. S1, which does not contain Mg2+ or other divalent cations.Limited proteolysis of β protein was also investigated using chymotrypsin, subtilisin, and thermolysin (Figs. 1B and supplemental Fig. S2). Although in general chymotrypsin cleaves at sites very different from trypsin (after aromatic or aliphatic residues instead of basic residues), the two give very similar cleavage patterns for β protein; that is, a stable N-terminal domain (residues 1–131) in the absence of DNA and a larger series of fragments (residues 1–184, 9–184, 13–184, and 36–184) in the presence of DNA. With subtilisin, a non-sequence-specific protease, β protein alone is digested into two similar fragments, an N-terminal domain (residues 1–134) and a longer fragment (residues 1–184), which in this case persists even in the absence of DNA. DNA does, however, change the cleavage pattern such that several new fragments (residues 1–177, 13–182, 20–182) are observed. Similarly with thermolysin, which cleaves before apolar residues, N-terminal (residues 1–138) and longer (1–177) fragments are observed for the protein alone, whereas DNA shifts the pattern to a series of intermediate fragments (residues 1–177, 8–177, and 12–177). Interestingly, with thermolysin a fragment consisting of nearly full-length β-protein (residues 1–258) is significantly more stable in the absence of DNA than in its presence, indicating that some sites within β protein are more accessible in the DNA complex than in the free protein.We have also investigated the trypsin digestion of β protein preparations that give distinct oligomeric forms (rings and filaments) as seen by electron microscopy (18Passy S.I. Yu X. Li Z. Radding C.M. Egelman E.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4279-4284Crossref PubMed Scopus (82) Google Scholar). For the small ring preparation with Mg2+ in the absence of DNA, only the N-terminal core domain (residues 1–134) is observed. For the large ring preparation in the presence of a 33-mer oligonucleotide, a small amount of the fragment corresponding to residues 1–177 is also observed. For the filament preparation formed in the presence of a heat-denatured fragment of ΦX174 dsDNA, increased amounts of full-length and fragments corresponding to residues 1–177 and 1–230 are observed, and the pattern is similar to that of the DNA complex with sequentially added 33-mer oligonucleotides described above. This analysis is consistent with the notion that in the DNA complex formed with sequentially added complementary oligonucleotides, β protein likely exists as short filaments. Using size-exclusion chromatography on Superdex S-200, our preparation of β protein in the absence of DNA elutes as a distinct peak corresponding to a large oligomer (566 kDa or ∼19 subunits), consistent with a ring (data not shown). The complex with sequentially added oligonucleotides elutes as a distinct peak with a larger apparent size. Similarly, with native acrylamide gel electrophoresis the complex of β protein with sequentially added oligonucleotides forms a distinct band that is of lower mobility (presumably larger size) than several bands formed by β protein alone (data not shown).Probing the DNA Binding Regions of β Protein by Biotin Modification and Mass Spectrometry—To further probe the structure of β protein and its interactions with DNA, we used a method involving mass spectrometric analysis of protein modified by biotinylation at lysine residues. NHS-biotin reacts efficiently and specifically with the primary ϵ-amino group of lysine side chains to become covalently attached via a stable amide linkage. The modification alters the pattern of pep" @default.
• W1978620556 created "2016-06-24" @default.
• W1978620556 creator A5002243266 @default.
• W1978620556 creator A5018038526 @default.
• W1978620556 creator A5030204501 @default.
• W1978620556 creator A5067691635 @default.
• W1978620556 creator A5082023270 @default.
• W1978620556 creator A5087856186 @default.
• W1978620556 date "2006-09-01" @default.
• W1978620556 modified "2023-10-10" @default.
• W1978620556 title "Domain Structure and DNA Binding Regions of β Protein from Bacteriophage λ" @default.
• W1978620556 cites W1496490740 @default.
• W1978620556 cites W1512370781 @default.
• W1978620556 cites W1520987654 @default.
• W1978620556 cites W1556520758 @default.
• W1978620556 cites W1582021807 @default.
• W1978620556 cites W1584920009 @default.
• W1978620556 cites W1600950270 @default.
• W1978620556 cites W1641310924 @default.
• W1978620556 cites W185329323 @default.
• W1978620556 cites W1882633455 @default.
• W1978620556 cites W1964507408 @default.
• W1978620556 cites W1978352901 @default.
• W1978620556 cites W2000379854 @default.
• W1978620556 cites W2005986251 @default.
• W1978620556 cites W2011700794 @default.
• W1978620556 cites W2021984369 @default.
• W1978620556 cites W2034880487 @default.
• W1978620556 cites W2039319572 @default.
• W1978620556 cites W2059608315 @default.
• W1978620556 cites W2067131659 @default.
• W1978620556 cites W2071444861 @default.
• W1978620556 cites W2074187513 @default.
• W1978620556 cites W2078252258 @default.
• W1978620556 cites W2086199106 @default.
• W1978620556 cites W2139095560 @default.
• W1978620556 cites W2141833703 @default.
• W1978620556 cites W2401652560 @default.
• W1978620556 doi "https://doi.org/10.1074/jbc.m512450200" @default.
• W1978620556 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/1950251" @default.
• W1978620556 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16820360" @default.
• W1978620556 hasPublicationYear "2006" @default.
• W1978620556 type Work @default.
• W1978620556 sameAs 1978620556 @default.
• W1978620556 citedByCount "20" @default.
• W1978620556 countsByYear W19786205562012 @default.
• W1978620556 countsByYear W19786205562013 @default.
• W1978620556 countsByYear W19786205562016 @default.
• W1978620556 countsByYear W19786205562017 @default.
• W1978620556 countsByYear W19786205562019 @default.
• W1978620556 countsByYear W19786205562020 @default.
• W1978620556 countsByYear W19786205562021 @default.
• W1978620556 countsByYear W19786205562022 @default.
• W1978620556 crossrefType "journal-article" @default.
• W1978620556 hasAuthorship W1978620556A5002243266 @default.
• W1978620556 hasAuthorship W1978620556A5018038526 @default.
• W1978620556 hasAuthorship W1978620556A5030204501 @default.
• W1978620556 hasAuthorship W1978620556A5067691635 @default.
• W1978620556 hasAuthorship W1978620556A5082023270 @default.
• W1978620556 hasAuthorship W1978620556A5087856186 @default.
• W1978620556 hasBestOaLocation W19786205561 @default.
• W1978620556 hasConcept C104317684 @default.
• W1978620556 hasConcept C107824862 @default.
• W1978620556 hasConcept C134306372 @default.
• W1978620556 hasConcept C185592680 @default.
• W1978620556 hasConcept C25166345 @default.
• W1978620556 hasConcept C2776441376 @default.
• W1978620556 hasConcept C33923547 @default.
• W1978620556 hasConcept C36503486 @default.
• W1978620556 hasConcept C54355233 @default.
• W1978620556 hasConcept C547475151 @default.
• W1978620556 hasConcept C552990157 @default.
• W1978620556 hasConcept C70721500 @default.
• W1978620556 hasConcept C86803240 @default.
• W1978620556 hasConceptScore W1978620556C104317684 @default.
• W1978620556 hasConceptScore W1978620556C107824862 @default.
• W1978620556 hasConceptScore W1978620556C134306372 @default.
• W1978620556 hasConceptScore W1978620556C185592680 @default.
• W1978620556 hasConceptScore W1978620556C25166345 @default.
• W1978620556 hasConceptScore W1978620556C2776441376 @default.
• W1978620556 hasConceptScore W1978620556C33923547 @default.
• W1978620556 hasConceptScore W1978620556C36503486 @default.
• W1978620556 hasConceptScore W1978620556C54355233 @default.
• W1978620556 hasConceptScore W1978620556C547475151 @default.
• W1978620556 hasConceptScore W1978620556C552990157 @default.
• W1978620556 hasConceptScore W1978620556C70721500 @default.
• W1978620556 hasConceptScore W1978620556C86803240 @default.
• W1978620556 hasIssue "35" @default.
• W1978620556 hasLocation W19786205561 @default.
• W1978620556 hasLocation W19786205562 @default.
• W1978620556 hasLocation W19786205563 @default.
• W1978620556 hasOpenAccess W1978620556 @default.
• W1978620556 hasPrimaryLocation W19786205561 @default.
• W1978620556 hasRelatedWork W1828691184 @default.
• W1978620556 hasRelatedWork W1903732681 @default.
• W1978620556 hasRelatedWork W1991523530 @default.
• W1978620556 hasRelatedWork W2002128513 @default.
• W1978620556 hasRelatedWork W2020824267 @default.

• next