Abstract
The McrBC restriction system has the ability to restrict DNA containing 5-hydroxymethylcytosine, N4-methylcytosine, and 5-methylcytosine at specific sequences. The mcrB gene produces two gene products. The complete mcrB open reading frame produces a 51-kDa protein (McrB(L)) and a 33-kDa protein (McrB(S)). The smaller McrB polypeptide is produced from an in-frame, internal translational start site in the mcrB gene. The McrB(S) sequence is identical to that of McrB(L) except that it lacks 161 amino acids present at the N-terminal end of the latter protein. It has been suggested that McrB(L) is the DNA binding restriction subunit. The function of McrB(S) is unknown, although there has been speculation that it plays some role in the modulation of McrBC restriction. Studies of the function of McrB(S) have been challenging since it is produced in frame with McrB(L). In this study, we tested the effects of underproduction (via antisense RNA) and overproduction (via gene dosage) of mcrBC gene products on restriction levels of the mcrBC+ strain JM107. Among the parameters monitored was the induction of SOS responses, which indicate of DNA damage. Evidence from this study suggests that McrB(S) is necessary for stabilization of the McrBC restriction complex in vivo.
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- Achberger E. C., Hilton M. D., Whiteley H. R. The effect of the delta subunit on the interaction of Bacillus subtilis RNA polymerase with bases in a SP82 early gene promoter. Nucleic Acids Res. 1982 May 11;10(9):2893–2910. doi: 10.1093/nar/10.9.2893. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
- Buhk H. J., Behrens B., Tailor R., Wilke K., Prada J. J., Günthert U., Noyer-Weidner M., Jentsch S., Trautner T. A. Restriction and modification in Bacillus subtilis: nucleotide sequence, functional organization and product of the DNA methyltransferase gene of bacteriophage SPR. Gene. 1984 Jul-Aug;29(1-2):51–61. doi: 10.1016/0378-1119(84)90165-3. [DOI] [PubMed] [Google Scholar]
- Castilho B. A., Olfson P., Casadaban M. J. Plasmid insertion mutagenesis and lac gene fusion with mini-mu bacteriophage transposons. J Bacteriol. 1984 May;158(2):488–495. doi: 10.1128/jb.158.2.488-495.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dila D., Raleigh E. A. Genetic dissection of the methylcytosine-specific restriction system mcrB of Escherichia coli K-12. Gene. 1988 Dec 25;74(1):23–24. doi: 10.1016/0378-1119(88)90241-7. [DOI] [PubMed] [Google Scholar]
- Dila D., Sutherland E., Moran L., Slatko B., Raleigh E. A. Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12. J Bacteriol. 1990 Sep;172(9):4888–4900. doi: 10.1128/jb.172.9.4888-4900.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dower W. J., Miller J. F., Ragsdale C. W. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res. 1988 Jul 11;16(13):6127–6145. doi: 10.1093/nar/16.13.6127. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Georgopoulos C. P. Isolation and preliminary characterization of T4 mutants with nonglucosylated DNA. Biochem Biophys Res Commun. 1967 Jul 21;28(2):179–184. doi: 10.1016/0006-291x(67)90426-3. [DOI] [PubMed] [Google Scholar]
- Kelleher J. E., Raleigh E. A. A novel activity in Escherichia coli K-12 that directs restriction of DNA modified at CG dinucleotides. J Bacteriol. 1991 Aug;173(16):5220–5223. doi: 10.1128/jb.173.16.5220-5223.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kiss A., Baldauf F. Molecular cloning and expression in Escherichia coli of two modification methylase genes of Bacillus subtilis. Gene. 1983 Jan-Feb;21(1-2):111–119. doi: 10.1016/0378-1119(83)90153-1. [DOI] [PubMed] [Google Scholar]
- Kiss A., Posfai G., Keller C. C., Venetianer P., Roberts R. J. Nucleotide sequence of the BsuRI restriction-modification system. Nucleic Acids Res. 1985 Sep 25;13(18):6403–6421. doi: 10.1093/nar/13.18.6403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kretz P. L., Kohler S. W., Short J. M. Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains. J Bacteriol. 1991 Aug;173(15):4707–4716. doi: 10.1128/jb.173.15.4707-4716.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krüger T., Grund C., Wild C., Noyer-Weidner M. Characterization of the mcrBC region of Escherichia coli K-12 wild-type and mutant strains. Gene. 1992 May 1;114(1):1–12. doi: 10.1016/0378-1119(92)90700-y. [DOI] [PubMed] [Google Scholar]
- Krüger T., Wild C., Noyer-Weidner M. McrB: a prokaryotic protein specifically recognizing DNA containing modified cytosine residues. EMBO J. 1995 Jun 1;14(11):2661–2669. doi: 10.1002/j.1460-2075.1995.tb07264.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- LURIA S. E., HUMAN M. L. A nonhereditary, host-induced variation of bacterial viruses. J Bacteriol. 1952 Oct;64(4):557–569. doi: 10.1128/jb.64.4.557-569.1952. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lederberg E. M., Cohen S. N. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. doi: 10.1128/jb.119.3.1072-1074.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Noyer-Weidner M. A novel mcrB-based Escherichia coli K-12 vector system and its use in analyzing the genetic determinants for the McrB nuclease. Gene. 1988 Dec 25;74(1):177–178. doi: 10.1016/0378-1119(88)90279-x. [DOI] [PubMed] [Google Scholar]
- Noyer-Weidner M., Diaz R., Reiners L. Cytosine-specific DNA modification interferes with plasmid establishment in Escherichia coli K12: involvement of rglB. Mol Gen Genet. 1986 Dec;205(3):469–475. doi: 10.1007/BF00338084. [DOI] [PubMed] [Google Scholar]
- Raleigh E. A., Murray N. E., Revel H., Blumenthal R. M., Westaway D., Reith A. D., Rigby P. W., Elhai J., Hanahan D. McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning. Nucleic Acids Res. 1988 Feb 25;16(4):1563–1575. doi: 10.1093/nar/16.4.1563. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raleigh E. A. Organization and function of the mcrBC genes of Escherichia coli K-12. Mol Microbiol. 1992 May;6(9):1079–1086. doi: 10.1111/j.1365-2958.1992.tb01546.x. [DOI] [PubMed] [Google Scholar]
- Raleigh E. A., Trimarchi R., Revel H. Genetic and physical mapping of the mcrA (rglA) and mcrB (rglB) loci of Escherichia coli K-12. Genetics. 1989 Jun;122(2):279–296. doi: 10.1093/genetics/122.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raleigh E. A., Wilson G. Escherichia coli K-12 restricts DNA containing 5-methylcytosine. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9070–9074. doi: 10.1073/pnas.83.23.9070. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Revel H. R. Restriction of nonglucosylated T-even bacteriophage: properties of permissive mutants of Escherichia coli B and K12. Virology. 1967 Apr;31(4):688–701. doi: 10.1016/0042-6822(67)90197-3. [DOI] [PubMed] [Google Scholar]
- Ross T. K., Achberger E. C., Braymer H. D. Characterization of the Escherichia coli modified cytosine restriction (mcrB) gene. Gene. 1987;61(3):277–289. doi: 10.1016/0378-1119(87)90191-0. [DOI] [PubMed] [Google Scholar]
- Ross T. K., Achberger E. C., Braymer H. D. Identification of a second polypeptide required for McrB restriction of 5-methylcytosine-containing DNA in Escherichia coli K12. Mol Gen Genet. 1989 Apr;216(2-3):402–407. doi: 10.1007/BF00334382. [DOI] [PubMed] [Google Scholar]
- Ross T. K., Achberger E. C., Braymer H. D. Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus. J Bacteriol. 1989 Apr;171(4):1974–1981. doi: 10.1128/jb.171.4.1974-1981.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanger F., Coulson A. R., Hong G. F., Hill D. F., Petersen G. B. Nucleotide sequence of bacteriophage lambda DNA. J Mol Biol. 1982 Dec 25;162(4):729–773. doi: 10.1016/0022-2836(82)90546-0. [DOI] [PubMed] [Google Scholar]
- Schoemaker J. M., Gayda R. C., Markovitz A. Regulation of cell division in Escherichia coli: SOS induction and cellular location of the sulA protein, a key to lon-associated filamentation and death. J Bacteriol. 1984 May;158(2):551–561. doi: 10.1128/jb.158.2.551-561.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sutherland E., Coe L., Raleigh E. A. McrBC: a multisubunit GTP-dependent restriction endonuclease. J Mol Biol. 1992 May 20;225(2):327–348. doi: 10.1016/0022-2836(92)90925-a. [DOI] [PubMed] [Google Scholar]
- Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
- Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
- Zheng L., Braymer H. D. Overproduction and purification of McrC protein from Escherichia coli K-12. J Bacteriol. 1991 Jun;173(12):3918–3920. doi: 10.1128/jb.173.12.3918-3920.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
