Illegitimate recombination at the replication origin of bacteriophage M13.

Abstract

Hybrids composed of phage M13 and plasmid pHV33 were used to study the formation of deletions in Escherchia coli. Eighty to ninety percent of the deletion endpoints were at the position of the nick introduced into the M13 replication origin by the phage gene II protein. This suggests the existence of a novel mechanism of illegitimate recombination.Recombination between sequences with little or no homology was termed "illegitimate" by Franklin (1) ref. 14), and AB1157 (thrAl leu-6 thi-) proA2 his4 argE3 lacYl galK2 ara-14 xyl-S mtl-i tsx-33 rpl-31 supE44; J.Clark, University of California, Berkeley). The Rec-strains were all isogenic to AB1157. They were rendered hsdR by conjugation with GY2098 (HfrH) after cotransfer of hsdR and thr'. recA strains JC5495 and JC5547 were rendered Rec' prior to conjugation by introduction of a RecAI X phage. This phage was eliminated from the exconjugants by segregation. All the Rec-strains harbored F'lacIqZ M15 pro' plasmid (15). The Rec-phenotype was routinely tested by sensitivity to ultraviolet light. The Rep-strain D162 was rendered hsdR by conjugation with a leu+ revertant of GY2098 after cotransfer of hsdR and leu+ markers. It harbored pOXKm plasmid (M. Chandler, Toulouse University).The plasmid pHV33 is composed of pBR322 and pC194 (16-18). The plasmids pHV672 (19) and pHV673 were constructed by joining EcoRI-cleaved M13 mp2 phage (20) to pHV33; they were isolated in B. subtilis. pHV698 was constructed by replacing the small pBR322 Cla I-EcoRI fragment with the 540-base-pair (bp) M13 mp2 Cla I-EcoRI fragment that carries the phage replication origin. pHV702 was obtained by inserting pC194 into the HindIII site of pHV698. R199 and R229 are two fl derivatives (21,22) (28,29) and was purified by chromatography on hydroxyapatite columns (30). For analytical purposes, plasmid DNA was extracted from 1.5-ml overnight cultures (31). The terminal-labeling method was used for DNA sequencing (32). Transformation of B. subtilis and E. coli competent cells and plasmid transduction have been described (28,33,34). RESULTSIsolation of Deletion Plasmids. Plasmid pHV33, composed ofthe E. coli plasmid pBR322 and the Staphylococcus aureus plasmid pC194, is viable in E. coli (18). We have joined the Abbreviations: bp, base pair(s); kb, kilobase(s). 3386The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Palindrome Regeneration by Template Strand-Switching Mechanism at the Origin of DNA Replication of Porcine Circovirus via the Rolling-Circle Melting-Pot Replication Model

“…In combination with one or more deletion events, the upstream sequence at positions l, m, n, o, and p or the downstream sequence at positions 24 to 30 can be recruited into the melting pot to generate vLD11.1, vLD11.2, vLD11.4, and vRD10, respectively. Ample examples of illegitimate recombination errors (i.e., deletions, insertions, and illegitimate nucleotide incorporation) that occur frequently at the origin of phages and plasmids (25,26) were also detected here. Insertions or duplications (e.g., vLD7.1, vRD3.2, and vRD3.5) and deletions (e.g., selected vLD7, selected vLD11, and vRD10) were observed among the progeny viruses.…”

Four DNA-A variants among Pakistani isolates of cotton leaf curl virus and their affinities to DNA-A of geminivirus isolates from okra.

“…This is plausible because ori is also a hot-spot for recombination during replication of the circular single-stranded DNA of phage M13 (Michel & Ehrlich, 1986) and was a site of forced recombination in African cassava mosaic virus (Stanley, 1995). How would the opportunity for recombination of these geminiviruses arise ?…”

Bacterial Genome Instability

“…Illegitimate recombination occurring during DNA synthesis can be responsible for local sequence conversion, deletion, or duplication. It can arise during DNA replication (336,337), transposition (91, 338), or gyrase-and topoisomerase I-mediated strand cleavage (339-341); as a consequence of UV or gamma irradiation (342-344); or following the transformation of cells with linear DNA sequences under circumstances where homologous recombination is not possible (345,346). Single-strand annealing following a DNA break can occur after DNA degradation, leading to a local deletion.…”