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. 1994 Dec 15;13(24):6152–6161. doi: 10.1002/j.1460-2075.1994.tb06962.x

Chromosome end formation in phage lambda, catalyzed by terminase, is controlled by two DNA elements of cos, cosN and R3, and by ATP.

R R Higgins 1, A Becker 1
PMCID: PMC395595  PMID: 7813452

Abstract

The terminase enzyme of phage lambda is a site-specific endonuclease that nicks DNA concatemers to regenerate the 12 nucleotide cohesive ends of the mature chromosome. The enzyme's DNA target, cos, consists of a nicking domain, cosN, and a binding domain, cosB. cosB, situated to the right of cosN, comprises three 16 bp repeat sequences, R1, R2 and R3. A similar sequence, R4, is present to the left of cosN. It is shown here that terminase has an intrinsic specificity for cosN which is independent of the R sites. The interaction with cosN is mediated by binding to target sites that include 12 bp on the 5', and 2-7 bp on the 3' side of the nick. Of the four R sites, only R3 is required for the proper formation of ends. When R3 is present, an ATP-charged terminase system correctly catalyzes the production of staggered nicks in cosN, at sites N1 and N2 on the bottom and top strands, respectively. When ATP is omitted, the bottom strand is nicked incorrectly, at the site Nx, 8 bp to the left of N1. If R3 is removed or disabled by a point mutation, nicking in cosN becomes dependent upon ATP but, even in the presence of ATP, bottom strand nicking is divided between sites N1, the correct site, and Nx, the incorrect one. Thus, R3 is an important regulatory element and must reside in cis in respect to cosN. Furthermore, cosN substrates bearing point mutations at N1 and N2 are nicked at sites Nx and Ny, 8 bp to the left of N1 and N2, respectively. When R3 is present and ATP is added, nicking is redirected to the N1 and N2 positions despite the mutations present. Thus, terminase binding to R3, on one side of cosN, regulates the rotationally symmetric nicking reactions on the bottom and top strands within cosN.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bear S. E., Court D. L., Friedman D. I. An accessory role for Escherichia coli integration host factor: characterization of a lambda mutant dependent upon integration host factor for DNA packaging. J Virol. 1984 Dec;52(3):966–972. doi: 10.1128/jvi.52.3.966-972.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Becker A., Gold M. Enzymatic breakage of the cohesive end site of phage lambda DNA: terminase (ter) reaction. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4199–4203. doi: 10.1073/pnas.75.9.4199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Becker A., Gold M. Prediction of an ATP reactive center in the small subunit, gpNu1, of the phage lambda terminase enzyme. J Mol Biol. 1988 Jan 5;199(1):219–222. doi: 10.1016/0022-2836(88)90391-9. [DOI] [PubMed] [Google Scholar]
  4. Becker A., Marko M., Gold M. Early events in the in vitro packaging of bacteriophage lambda DNA. Virology. 1977 May 1;78(1):291–305. doi: 10.1016/0042-6822(77)90100-3. [DOI] [PubMed] [Google Scholar]
  5. Becker A., Murialdo H. Bacteriophage lambda DNA: the beginning of the end. J Bacteriol. 1990 Jun;172(6):2819–2824. doi: 10.1128/jb.172.6.2819-2824.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brennan R. G., Matthews B. W. The helix-turn-helix DNA binding motif. J Biol Chem. 1989 Feb 5;264(4):1903–1906. [PubMed] [Google Scholar]
  7. Cue D., Feiss M. A site required for termination of packaging of the phage lambda chromosome. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9290–9294. doi: 10.1073/pnas.90.20.9290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cue D., Feiss M. Genetic analysis of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda. J Mol Biol. 1992 Nov 5;228(1):58–71. doi: 10.1016/0022-2836(92)90491-2. [DOI] [PubMed] [Google Scholar]
  9. Cue D., Feiss M. Genetic analysis of mutations affecting terminase, the bacteriophage lambda DNA packaging enzyme, that suppress mutations in cosB, the terminase binding site. J Mol Biol. 1992 Nov 5;228(1):72–87. doi: 10.1016/0022-2836(92)90492-3. [DOI] [PubMed] [Google Scholar]
  10. Davidson A. R., Gold M. Mutations abolishing the endonuclease activity of bacteriophage lambda terminase lie in two distinct regions of the A gene, one of which may encode a "leucine zipper" DNA-binding domain. Virology. 1992 Jul;189(1):21–30. doi: 10.1016/0042-6822(92)90677-h. [DOI] [PubMed] [Google Scholar]
  11. Delarue M., Poch O., Tordo N., Moras D., Argos P. An attempt to unify the structure of polymerases. Protein Eng. 1990 May;3(6):461–467. doi: 10.1093/protein/3.6.461. [DOI] [PubMed] [Google Scholar]
  12. Ellenberger T. E., Brandl C. J., Struhl K., Harrison S. C. The GCN4 basic region leucine zipper binds DNA as a dimer of uninterrupted alpha helices: crystal structure of the protein-DNA complex. Cell. 1992 Dec 24;71(7):1223–1237. doi: 10.1016/s0092-8674(05)80070-4. [DOI] [PubMed] [Google Scholar]
  13. Feiss M., Fogarty S., Christiansen S. Bacteriophage lambda DNA packaging: a mutant terminase that is independent of integration host factor. Mol Gen Genet. 1988 Apr;212(1):142–148. doi: 10.1007/BF00322457. [DOI] [PubMed] [Google Scholar]
  14. Feiss M., Frackman S., Sippy J. Essential interaction between lambdoid phage 21 terminase and the Escherichia coli integrative host factor. J Mol Biol. 1985 May 25;183(2):239–246. doi: 10.1016/0022-2836(85)90216-5. [DOI] [PubMed] [Google Scholar]
  15. Feiss M., Kobayashi I., Widner W. Separate sites for binding and nicking of bacteriophage lambda DNA by terminase. Proc Natl Acad Sci U S A. 1983 Feb;80(4):955–959. doi: 10.1073/pnas.80.4.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Feiss M., Widner W., Miller G., Johnson G., Christiansen S. Structure of the bacteriophage lambda cohesive end site: location of the sites of terminase binding (cosB) and nicking (cosN). Gene. 1983 Oct;24(2-3):207–218. doi: 10.1016/0378-1119(83)90081-1. [DOI] [PubMed] [Google Scholar]
  17. Frackman S., Siegele D. A., Feiss M. A functional domain of bacteriophage lambda terminase for prohead binding. J Mol Biol. 1984 Dec 5;180(2):283–300. doi: 10.1016/s0022-2836(84)80005-4. [DOI] [PubMed] [Google Scholar]
  18. Frackman S., Siegele D. A., Feiss M. The terminase of bacteriophage lambda. Functional domains for cosB binding and multimer assembly. J Mol Biol. 1985 May 25;183(2):225–238. doi: 10.1016/0022-2836(85)90215-3. [DOI] [PubMed] [Google Scholar]
  19. Gold M., Becker A. The bacteriophage lambda terminase. Partial purification and preliminary characterization of properties. J Biol Chem. 1983 Dec 10;258(23):14619–14625. [PubMed] [Google Scholar]
  20. Granston A. E., Alessi D. M., Eades L. J., Friedman D. I. A point mutation in the Nul gene of bacteriophage lambda facilitates phage growth in Escherichia coli with himA and gyrB mutations. Mol Gen Genet. 1988 Apr;212(1):149–156. doi: 10.1007/BF00322458. [DOI] [PubMed] [Google Scholar]
  21. Guo P., Peterson C., Anderson D. Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gp16 of bacteriophage phi 29. J Mol Biol. 1987 Sep 20;197(2):229–236. doi: 10.1016/0022-2836(87)90121-5. [DOI] [PubMed] [Google Scholar]
  22. Higgins R. R., Becker A. The lambda terminase enzyme measures the point of its endonucleolytic attack 47 +/- 2 bp away from its site of specific DNA binding, the R site. EMBO J. 1994 Dec 15;13(24):6162–6171. doi: 10.1002/j.1460-2075.1994.tb06963.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Higgins R. R., Lucko H. J., Becker A. Mechanism of cos DNA cleavage by bacteriophage lambda terminase: multiple roles of ATP. Cell. 1988 Sep 9;54(6):765–775. doi: 10.1016/s0092-8674(88)91021-5. [DOI] [PubMed] [Google Scholar]
  24. Kypr J., Mrázek J. Lambda phage protein Nu 1 contains the conserved DNA binding fold of repressors. J Mol Biol. 1986 Sep 5;191(1):139–140. doi: 10.1016/0022-2836(86)90430-4. [DOI] [PubMed] [Google Scholar]
  25. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  26. McClarin J. A., Frederick C. A., Wang B. C., Greene P., Boyer H. W., Grable J., Rosenberg J. M. Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution. Science. 1986 Dec 19;234(4783):1526–1541. doi: 10.1126/science.3024321. [DOI] [PubMed] [Google Scholar]
  27. Miller G., Feiss M. The bacteriophage lambda cohesive end site: isolation of spacing/substitution mutations that result in dependence on Escherichia coli integration host factor. Mol Gen Genet. 1988 Apr;212(1):157–165. doi: 10.1007/BF00322459. [DOI] [PubMed] [Google Scholar]
  28. Miwa T., Matsubara K. Lambda phage DNA sequences affecting the packaging process. Gene. 1983 Oct;24(2-3):199–206. doi: 10.1016/0378-1119(83)90080-x. [DOI] [PubMed] [Google Scholar]
  29. Parris W., Davidson A., Keeler C. L., Jr, Gold M. The Nu1 subunit of bacteriophage lambda terminase. J Biol Chem. 1988 Jun 15;263(17):8413–8419. [PubMed] [Google Scholar]
  30. Shinder G., Gold M. The Nul subunit of bacteriophage lambda terminase binds to specific sites in cos DNA. J Virol. 1988 Feb;62(2):387–392. doi: 10.1128/jvi.62.2.387-392.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sumner-Smith M., Becker A. DNA packaging in the lambdoid phages: identification of the products of phi 80 genes 1 and 2. Virology. 1981 Jun;111(2):629–641. doi: 10.1016/0042-6822(81)90362-7. [DOI] [PubMed] [Google Scholar]
  32. Sumner-Smith M., Becker A., Gold M. DNA packaging in the lambdoid phages: the role of lambda genes Nu1 and A. Virology. 1981 Jun;111(2):642–646. doi: 10.1016/0042-6822(81)90363-9. [DOI] [PubMed] [Google Scholar]
  33. Szybalski W., Kim S. C., Hasan N., Podhajska A. J. Class-IIS restriction enzymes--a review. Gene. 1991 Apr;100:13–26. doi: 10.1016/0378-1119(91)90345-c. [DOI] [PubMed] [Google Scholar]
  34. Tomka M. A., Catalano C. E. Physical and kinetic characterization of the DNA packaging enzyme from bacteriophage lambda. J Biol Chem. 1993 Feb 15;268(5):3056–3065. [PubMed] [Google Scholar]
  35. Vinson C. R., Sigler P. B., McKnight S. L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science. 1989 Nov 17;246(4932):911–916. doi: 10.1126/science.2683088. [DOI] [PubMed] [Google Scholar]
  36. Weigel P. H., Englund P. T., Murray K., Old R. W. The 3'-terminal nucleotide sequences of bacteriophage lambda DNA. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1151–1155. doi: 10.1073/pnas.70.4.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Xin W. N., Feiss M. The interaction of Escherichia coli integration host factor with the cohesive end sites of phages lambda and 21. Nucleic Acids Res. 1988 Mar 25;16(5):2015–2030. doi: 10.1093/nar/16.5.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Xu S. Y., Feiss M. Structure of the bacteriophage lambda cohesive end site. Genetic analysis of the site (cosN) at which nicks are introduced by terminase. J Mol Biol. 1991 Jul 20;220(2):281–292. doi: 10.1016/0022-2836(91)90013-v. [DOI] [PubMed] [Google Scholar]

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