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. 1990 Aug 1;269(3):679–684. doi: 10.1042/bj2690679

Purification of overexpressed gam gene protein from bacteriophage Mu by denaturation-renaturation techniques and a study of its DNA-binding properties.

Z H Abraham 1, N Symonds 1
PMCID: PMC1131641  PMID: 2167662

Abstract

Recombinant Mu gam gene protein (Mu GAM) synthesized in Escherichia coli accumulates in the form of insoluble inclusion bodies which, after cell lysis and low-speed centrifugation, can be recovered in the pellet fraction. This property was utilized in a purification procedure for Mu GAM based on guanidine hydrochloride denaturation-renaturation followed by a single DEAE-cellulose chromatographic step. The purified Mu GAM was shown by nitrocellulose-filter-binding experiments to bind with high affinity to linear double-stranded DNA and more weakly to supercoiled and single-stranded forms. Mu GAM protects linear DNA from degradation by a variety of exonucleases, but only weakly inhibits endonuclease activity. These results are in accord with a model of Mu GAM conferring protection from exonuclease activity by binding to the ends of DNA.

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Selected References

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

  1. Akroyd J. E., Clayson E., Higgins N. P. Purification of the gam gene-product of bacteriophage Mu and determination of the nucleotide sequence of the gam gene. Nucleic Acids Res. 1986 Sep 11;14(17):6901–6914. doi: 10.1093/nar/14.17.6901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akroyd J., Barton B., Lund P., Maynard Smith S., Sultana K., Symonds N. Mapping and properties of the gam and sot genes of phage mu: their possible roles in recombination. Cold Spring Harb Symp Quant Biol. 1984;49:261–266. doi: 10.1101/sqb.1984.049.01.030. [DOI] [PubMed] [Google Scholar]
  3. Akroyd J., Symonds N. Localization of the gam gene of bacteriophage mu and characterisation of the gene product. Gene. 1986;49(2):273–282. doi: 10.1016/0378-1119(86)90288-x. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Enquist L. W., Skalka A. Replication of bacteriophage lambda DNA dependent on the function of host and viral genes. I. Interaction of red, gam and rec. J Mol Biol. 1973 Apr 5;75(2):185–212. doi: 10.1016/0022-2836(73)90016-8. [DOI] [PubMed] [Google Scholar]
  6. Gloor G., Chaconas G. The bacteriophage Mu N gene encodes the 64-kDa virion protein which is injected with, and circularizes, infecting Mu DNA. J Biol Chem. 1986 Dec 15;261(35):16682–16688. [PubMed] [Google Scholar]
  7. Gribskov M., Burgess R. R. Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase. Gene. 1983 Dec;26(2-3):109–118. doi: 10.1016/0378-1119(83)90180-4. [DOI] [PubMed] [Google Scholar]
  8. Harshey R. M., Bukhari A. I. Infecting bacteriophage mu DNA forms a circular DNA-protein complex. J Mol Biol. 1983 Jun 25;167(2):427–441. doi: 10.1016/s0022-2836(83)80343-x. [DOI] [PubMed] [Google Scholar]
  9. Hsieh T., Brutlag D. L. A protein that preferentially binds Drosophila satellite DNA. Proc Natl Acad Sci U S A. 1979 Feb;76(2):726–730. doi: 10.1073/pnas.76.2.726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnson A. D., Pabo C. O., Sauer R. T. Bacteriophage lambda repressor and cro protein: interactions with operator DNA. Methods Enzymol. 1980;65(1):839–856. doi: 10.1016/s0076-6879(80)65078-2. [DOI] [PubMed] [Google Scholar]
  11. Karu A. E., Sakaki Y., Echols H., Linn S. The gamma protein specified by bacteriophage gamma. Structure and inhibitory activity for the recBC enzyme of Escherichia coli. J Biol Chem. 1975 Sep 25;250(18):7377–7387. [PubMed] [Google Scholar]
  12. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  13. Marston F. A. The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J. 1986 Nov 15;240(1):1–12. doi: 10.1042/bj2400001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. McEntee K., Weinstock G. M., Lehman I. R. Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2615–2619. doi: 10.1073/pnas.76.6.2615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Poteete A. R., Fenton A. C., Murphy K. C. Modulation of Escherichia coli RecBCD activity by the bacteriophage lambda Gam and P22 Abc functions. J Bacteriol. 1988 May;170(5):2012–2021. doi: 10.1128/jb.170.5.2012-2021.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sakaki Y. Inactivation of the ATP-dependent DNase of Escherichia coli after infection with double-stranded DNA phages. J Virol. 1974 Dec;14(6):1611–1612. doi: 10.1128/jvi.14.6.1611-1612.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schaus N. A., Wright A. Inhibition of Escherichia coli exonuclease V by bacteriophage Mu. Virology. 1980 Apr 15;102(1):214–217. doi: 10.1016/0042-6822(80)90083-5. [DOI] [PubMed] [Google Scholar]
  18. Silverstein J. L., Goldberg E. B. T4 DNA injection. II. Protection of entering DNA from host exonuclease V. Virology. 1976 Jul 1;72(1):212–223. doi: 10.1016/0042-6822(76)90324-x. [DOI] [PubMed] [Google Scholar]
  19. Smolarsky M., Tal M. Novel method for measuring polyuridylic acid binding to ribosomes. Biochim Biophys Acta. 1970 Feb 18;199(2):447–452. doi: 10.1016/0005-2787(70)90087-0. [DOI] [PubMed] [Google Scholar]
  20. Surette M. G., Buch S. J., Chaconas G. Transpososomes: stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNA. Cell. 1987 Apr 24;49(2):253–262. doi: 10.1016/0092-8674(87)90566-6. [DOI] [PubMed] [Google Scholar]
  21. Van Vliet F., Couturier M., De Lafonteyne J., Jedlicki E. Mu-1 directed inhibition of DNA breakdown in Escherichia coli, recA cells. Mol Gen Genet. 1978 Aug 4;164(1):109–112. doi: 10.1007/BF00267606. [DOI] [PubMed] [Google Scholar]
  22. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

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