Lambda red-mediated synthesis of plasmid linear multimers in Escherichia coli K12

Abstract

Expression of the red+ and gam+ genes of bacteriophage lambda in plasmids cloned in Escherichia coli wild-type cells leads to plasmid linear multimer (PLM) formation. In mutants that lack exonuclease I (sbcB sbcC), either of these lambda functions mediates PLM formation. In order to determine whether PLM formation in sbcB sbcC mutants occurs by conservative (break-join) recombination of circular plasmids or by de novo DNA synthesis, thyA sbcB sbcC mutants were transferred from thymine- to 5-bromo-2'-deoxyuridine (BUDR)-supplemented medium, concurrently with induction of red+ or gam+ expression, and the density distribution of plasmid molecular species was analyzed. After a period of less than one generation in the BUDR-supplemented medium, most PLM were of heavy/heavy density. Circular plasmids, as well as chromosomal DNA, were of light/light or light/heavy density. These results indicate that Red or Gam activities mediate de novo synthesis of PLM in sbcB sbcC mutants. Examination of plasmid DNA preparations from sbcB sbcC mutants expressing gam+ or red+ reveals the presence of two molecular species that may represent intermediates in the PLM biosynthesis pathway: single-branched circles (sigma-structures) and PLM with single-stranded DNA tails. While Gam-mediated PLM synthesis in sbcB mutants depends on the activity of the RecF pathway genes, Red-mediated PLM synthesis, like Red-mediated recombination, is independent of recA and recF activities. One of the red+ products, beta protein, suppresses RecA deficiency in plasmid recombination and PLM synthesis in RecBCD- ExoI- cells. The dependence of PLM synthesis on the RecE, RecF or Red recombination pathways and the dependence of plasmid recombination by these pathways on activities that are required for plasmid replication support the proposal that PLM synthesis and recombination by these pathways are mutually dependent. We propose the hypothesis that DNA double-stranded ends, which are produced in the process of PLM synthesis, are involved in plasmid recombination by the RecE, RecF and Red pathways. Conversely, recombination-dependent priming of DNA synthesis at 3' single-stranded DNA ends is hypothesized to initiate PLM synthesis on circular plasmid DNA templates.

λ Recombination and Recombineering

“…It is noteworthy that these DNA forms are circular dimers, trimers, and tetramers containing mixed populations of parental and recombinant markers, and are not the same as the very large linear multimers observed with colE1 plasmids replicating in a host constitutive for Red, Gam, or Red + Gam expression (140,262,263). The plasmid multimers were not observed in cells induced for Red + Gam in the absence of added DNA.…”

Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

“…It was initially proposed that Mhr1 may have a Rad52-type activity like Red␤ in the phage . Red␤ is known to initiate rolling-circle replication by annealing the Red␤-ssDNA filaments preferentially on the lagging strand of a replication fork, followed by template switch and DNA synthesis (172,173,203). The recent finding of Mgm101 as a Rad52-type protein raises the possibility that Mgm101 may fulfill such a role, in a manner dependent on or independent of Mhr1 (146,167).…”

DNA Recombination-Initiation Plays a Role in the Extremely Biased Inheritance of Yeast [rho−] Mitochondrial DNA That Contains the Replication Origin ori5

“…Inspired by the late-stage replication of the Escherichia coli phage (16,47), the recombination-mediated mechanism for the initiation of mtDNA replication in a rolling-circle mode has been proposed to explain mtDNA concatemer formation (37).…”