Studies of the recognition sequence of φX174 gene A protein. Cleavage site of φX gene A protein in St-1 RFI DNA

volume 8 Numb«r 91980 Nucleic Acids Research Studies of the recognition sequence of </>Xl 74 gene A protein. Cleavage site of tpX gene A protein in St-1 RFI DNA F.Heidekamp1 -2, S.A-Langeveld1 -3, P.D.Baas1 -2 and H.S.Jansz1 >2 Institute of Molecular Biology, Laboratory for Physiological Chemistry, and J Department of Molecular Cell Biology, State University of Utrecht, Utrecht, Netherlands Received 29 January 1980 It is already known that (JX gene A protein converts besides OX RFI DNA also the RFI DNAs o f the single-stranded bacteriophages G't, S t - 1 , a 3 and 0 K into RFII DNA. W e have extended this observation for bacteriophages Glk and U3• Restriction enzyme analysis placed the 0X gene A protein cleavage site in St-1 RF DNA in the Hinf\ restriction DNA fragment F I Q and in the overlapping ffaelll restriction DNA fragment Zj. The exact position and the nucleotide sequence at the 3'-0H end o f the nick were determined by DNA sequence analysis of the single-stranded DNA subfragment of the nicked DNA fragment Fio obtained by gelelectrophoresis in denaturing conditions. A s-tretch of 8 5 nucleotides o f St-1 DNA around the position o f the (JX gene A protein cleavage site was established by DNA sequence analysis o f the restriction DNA fragment Z7F1. Comparison o f this nucleotide sequence with the previously determined nucleotide sequence around the cleavage site of 0X gene A protein in 0X17*1 RF DNA and G1) RF DNA revealed an identical sequence o f only 10 nucleotides. The results suggest that the recognition sequence o f the 0X17 1 * gene A protein lies within these 10 nucleotides. INTRODUCTION Bacteriophage 0X17** gene A protein is the only phage-coded protein required for (JX RF DNA replication, as was first shown by Tessman (1) for the closely related bacteriophage S13. Initiation o f 0X RF DNA replication is mediated by the endonucleoiytic action o f the gene A protein, which introduces a single nick in the viral strand o f 0X RFI DNA ( 2 ) . This nick serves as the origin of (JX RF DNA repl i cat ion. The origin o f 0 X RF DNA replication has been placed by in vivo (3) and in vitro studies Ct,5) in a particular region o f the Hae\\\ restriction DNA fragment Zgg which is located within the viral gene A o f the 0X g e n o m e . The exact position of the (JX gene A protein cleavage site in 0X RF DNA has been determined by Langeveld et at. ( 6 ) . Their results indicate that 0X gene A protein cleaves the 3' sugai—phosphate bond o f the guanylic acid residue which corresponds with nucleotide 1*305 (7) In the viral strand o f 0X RFI DNA. © IRL Press United, 1 Falconberg Court, London W 1 V 5FG, U.K. 2009 ABSTRACT Nucleic Acids Research MATERIALS AND METHODS Preparation of RF DNA Escherichia coli was grown in a medium containing 10 g Bacto-Tryptone, 5 g KC1, 10 mM MgSOZ, and 2 mM CaCl2 per liter. As a host for St-1 and U3 Escherichia col! W3110 was used and for 0X17^, a3 and G14 E. coli BTCC 122. At a cell Q density of about 3 x 10 /ml the culture was infected with phage at a multiplicity of infection of 3-k. Chloramphenicol was added 2 min after infection to a concentration of kO pg/ml. RF DNA was prepared according to the method of Jansz et al. (12). Pure RFI DNA was isolate'd by CsCl buoyant density centrlfugatlon in the presence of 200 ug/ml ethidium bromide (13)Enzymes gene A protein was purified by affinity chroma tography using singlestranded DNA cellulose and Sepharose-heparin columns and showed no detectable contaminating protein bands as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Details of the purification procedure will be published elsewhere. The restriction endonucleases Hae\\\ and Hinf\ were isolated according to a combination of the methods of Takanami and Kojo O ' O and Takanami (15) as described by Vereijken (16). T^ polynucleotide kinase and the restriction endonuclease Alu\ were purchased from Boehringer (Mannheim); bacterial alkaline phosphatase (BAPF) was from Worthington (Freehold, N J ) , proteinase K was from Merck (Darmstadt). Restriction endonuclease digestions 2010 In studies on the recognition sequence of the SIX gene A protein, van Mansfeld et al. (8) have shown that RFI DNA of the 0X related bacter iophage G1) is also nicked at a unique site by incubation with 0X gene A protein. The SIX gene A protein cleaves the 3' sugai—phosphate bond of the guanylic acid residue which corresponds with nucleotide 506 (9) in the viral strand of Gh RF I DNA. Comparison of the viral strand DNA sequences around the 0X gene A protein cleavage sites In 0X RF DNA and G^ RF DNA revealed a stretch of 30 completely conserved nucleotides (8, 1 0 ) . In order to determine which nucleotides within that region are essential for the origin function we have studied the action of 0X gene A protein on RFI DNAs of other single-stranded DNA containing bacteriophages. The results, presented in this paper, confirm that the RFI DNAs of bacterlophages St-1 and a3 are also nicked by 0X gene A protein (11) and extend this observation for bacteriophages G14 and U3- The DNA sequence around the cleavage site in St-1 RF DNA has been established and compared with the corresponding sequences in 0X and G4. Nucleic Acids Research were performed 6.6 in a buffer mM B - m e r c a p t o ethanol containing 6 . 6 mM T r i s - H C l ( p H 7 . 5 ) . 6 . 6 mM a n d 5 0 mM N a C l . Chemicals 0X gene A protein incubation RFI DNA (0.5 yg) wes incubated with gene A protein (0.08 yg) in a reaction mixture of 25 yl containing 50 mM Tris-HCl (pH 7 - 6 ) , 10 mM MgCl 2 , 5 mM dithiothreitol, 1 mM E0TA and 120 mM NaCl for 30 min at 37 C. In control experiments, no gene A protein was added to the mixture. The reaction was terminated by adding EDTA to k§ mM and 50 yg proteinase K (pre incubated for 30 min at 37°C). The incubation was continued for 30 min at 37 C and then the conversion of RFI DNA into RFII DNA was analyzed by electrophoresis for 2.5 hrs at 150 Volts on horizontal \% agarose slab gels, containing 1 yg/ml ethidium bromide (17). Preparation and analysis of restriction endonuclease DNA fragments For preparative purposes, 30 yg St-1 RFI DNA was incubated with 2.6 yg 0X gene A protein in a reaction volume of 2.0 ml (composition as described above) for 90 min at 37 C. The reaction was terminated by adding EDTA to '(O mM and proteinase K (preincubated for 60 min at 37 C) to 300 yg/ml. The incubation was continued for 60 min at 37 C and followed by extraction with phenol. After ethanol precipitation the DNA was dissolved in 80 yl restriction enzyme buffer and an appropriate amount of restriction enzyme was added. The mixture was incubated for 2 hrs at 37 C and the reaction was terminated by adding EDTA to 10 mM. After phenol extraction, the DNA fragments were precipitated with ethanol and redissolved in 80 yl 50 mM Tris-HCl (pH 8 . 0 ) . The restriction DNA fragments were treated with alkaline phosphatase and labelled 1 1 at their 5 (...truncated)