Stability of DNA repeats in Escherichia coli dam mutant strains indicates a Dam methylation-dependent DNA deletion process

• Published in: Gene Vol. 258; no. 1; pp. 95 - 108
• Main Authors: Troester, Helmut, Bub, Sabine, Hunziker, Andreas, Trendelenburg, Michael F
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 27-11-2000
• Subjects: Base Sequence; Binding Sites - genetics; Cloning; DNA Methylation; DNA Restriction Enzymes - genetics; DNA Restriction Enzymes - metabolism; DNA Restriction-Modification Enzymes - genetics; DNA Restriction-Modification Enzymes - metabolism; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; mismatch repair; Molecular Sequence Data; Mutation; Plasmid; Plasmids - genetics; Rec A Recombinases - genetics; Rec A Recombinases - metabolism; RecA protein; Recombination, Genetic; Repetitive Sequences, Nucleic Acid; Sequence Deletion - genetics; Sequence Homology, Nucleic Acid; Short direct DNA repeat; Site-Specific DNA-Methyltransferase (Adenine-Specific) - genetics; Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism; Stabilization of repetitive DNA; Transformation, Bacterial; Sm, streptomycin; EDTA, ethylenediaminetetraacetic acid; seqA, gene encoding SeqA involved in the regulation of E. coli chromosomal replication; MCS, multiple cloning site(s); Cm, chloramphenicol; novel junction (fusion or insertion); kb, kilobase(s) or 1000 bp; C, cytidine; T7, T7 RNA promoter sequence; LB, Luria–Bertani; denotes plasmid-carrier state; R, resistance/resistant; Δ, deletion; hsd, genes encoding proteins involved in methylation-dependent restriction; wt, wild type; Oc, ochre suppressor mutation; oriC, ori of the E. coli chromosome; u, unit(s); mrr, gene encoding a protein involved in methylation-dependent restriction; recA, gene encoding RecA protein; T, thymidine; ori, origin(s) of replication; mut, genes encoding proteins involved in DNA repair; X, -fold or abbreviation of XL1-Blue; SK, SK primer DNA sequence element of the pBl MCS usually serving as annealing site for complementary sequencing oligos; mcr, genes encoding proteins involved in methylation-dependent restriction; Bl, Bluescript; dam, gene encoding Dam; Ap, ampicillin; mutHLS system, proteins MutH, MutL and MutS co-operatively; Dam, DNA adenine MTase; (+) indicates orientation of the f1 origin in phagemid-type plasmids; MTase, DNA methyltransferase; Tc, tetracycline; T3, T3 RNA promoter sequence; Stabilization of repetitive DNA; KS, orientation of the pBl MCS flanked by KpnI (K) and SacI (S); oligo, oligodeoxyribonucleotide; Short direct DNA repeat; bp, base pair(s); A, adenosine; G, guanosine; Tn, transposon; Tris, Tris (hydroxymethyl)-aminomethane; E. coli, Escherichia coli K-12; Cloning; Km, kanamycin; p, plasmid; Plasmid; methyl-DMR, methyl-directed mismatch repair
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(00)00420-0

In this study we report on the stabilization of short direct repetitive DNA elements. We arranged a 20
bp SK-primer element in a direct repeat manner within the cloning vector pBluescript KS (+). This resulted in an array of 27 direct repeats consisting of 24
bp units. We show that this plasmid could only be propagated without deletion of repeats in
dam mutant
Escherichia coli hosts, whereas all efforts to use strains that were defective in the methylation-dependent restriction system and the
recA- or the mismatch repair-dependent deletion system failed. The deletions always affected whole repeat units and not parts of them, leading to an unpredictable reduction of the unit number down to a range of between 12 and two during propagation. We conclude that a Dam methylation-dependent, but
recA- and mismatch repair-independent, deletion mechanism caused the DNA rearrangements without an obvious involvement of the known methylated-DNA restriction systems.