Homology Requirements for Double-Strand Break-Mediated Recombination in a Phage λ-td Intron Model System

Genetics ◽  
1996 ◽  
Vol 143 (3) ◽  
pp. 1057-1068 ◽  
Author(s):  
Monica M Parker ◽  
Deborah A Court ◽  
Karen Preiter ◽  
Marlene Belfort
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Model System ◽  
Group I Introns ◽  
Wild Type ◽  
Intron Insertion ◽  
Homologous Recombination Event ◽  
Group I ◽  
Intron Homing

Abstract Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage λ model system was developed to analyze exon homology effects on intron homing and determine the role of the λ 5′–3′ exonuclease complex (Redαβ) in the repair event. Efficient intron homing depended on exon lengths in the 35- to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type λ, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology.

scholarly journals Interaction of the intron-encoded mobility endonuclease I-PpoI with its target site.

1993 ◽  
Vol 13 (12) ◽  
pp. 7531-7539 ◽  
Author(s):  
E L Ellison ◽  
V M Vogt
Keyword(s):  
Gel Filtration ◽  
Dimethyl Sulfate ◽  
Strand Break ◽  
Target Site ◽  
Nuclear Ribosomal Dna ◽  
Group I Introns ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Intron Insertion ◽  
Group I

Endonucleases encoded by mobile group I introns are highly specific DNases that induce a double-strand break near the site to which the intron moves. I-PpoI from the acellular slime mold Physarum polycephalum mediates the mobility of intron 3 (Pp LSU 3) in the extrachromosomal nuclear ribosomal DNA of this organism. We showed previously that cleavage by I-PpoI creates a four-base staggered cut near the point of intron insertion. We have now characterized several further properties of the endonuclease. As determined by deletion analysis, the minimal target site recognized by I-PopI was a sequence of 13 to 15 bp spanning the cleavage site. The purified protein behaved as a globular dimer in sedimentation and gel filtration. In gel mobility shift assays in the presence of EDTA, I-PpoI formed a stable and specific complex with DNA, dissociating with a half-life of 45 min. By footprinting and interference assays with methidiumpropyl-EDTA-iron(II), I-PpoI contacted a 22- to 24-bp stretch of DNA. The endonuclease protected most of the purines found in both the major and minor grooves of the DNA helix from modification by dimethyl sulfate (DMS). However, the reactivity to DMS was enhanced at some purines, suggesting that binding leads to a conformational change in the DNA. The pattern of DMS protection differed fundamentally in the two partially symmetrical halves of the recognition sequence.

Interaction of the intron-encoded mobility endonuclease I-PpoI with its target site

1993 ◽  
Vol 13 (12) ◽  
pp. 7531-7539
Author(s):  
E L Ellison ◽  
V M Vogt
Keyword(s):  
Gel Filtration ◽  
Dimethyl Sulfate ◽  
Strand Break ◽  
Target Site ◽  
Nuclear Ribosomal Dna ◽  
Group I Introns ◽  
Recognition Sequence ◽  
Mobility Shift ◽  
Intron Insertion ◽  
Group I

Endonucleases encoded by mobile group I introns are highly specific DNases that induce a double-strand break near the site to which the intron moves. I-PpoI from the acellular slime mold Physarum polycephalum mediates the mobility of intron 3 (Pp LSU 3) in the extrachromosomal nuclear ribosomal DNA of this organism. We showed previously that cleavage by I-PpoI creates a four-base staggered cut near the point of intron insertion. We have now characterized several further properties of the endonuclease. As determined by deletion analysis, the minimal target site recognized by I-PopI was a sequence of 13 to 15 bp spanning the cleavage site. The purified protein behaved as a globular dimer in sedimentation and gel filtration. In gel mobility shift assays in the presence of EDTA, I-PpoI formed a stable and specific complex with DNA, dissociating with a half-life of 45 min. By footprinting and interference assays with methidiumpropyl-EDTA-iron(II), I-PpoI contacted a 22- to 24-bp stretch of DNA. The endonuclease protected most of the purines found in both the major and minor grooves of the DNA helix from modification by dimethyl sulfate (DMS). However, the reactivity to DMS was enhanced at some purines, suggesting that binding leads to a conformational change in the DNA. The pattern of DMS protection differed fundamentally in the two partially symmetrical halves of the recognition sequence.

scholarly journals Group I Intron Homing in Bacillus Phages SPO1 and SP82: a Gene Conversion Event Initiated by a Nicking Homing Endonuclease

2004 ◽  
Vol 186 (13) ◽  
pp. 4307-4314 ◽  
Author(s):  
Markus Landthaler ◽  
Nelson C. Lau ◽  
David. A. Shub
Keyword(s):  
Gene Conversion ◽  
Recombination Event ◽  
Homing Endonuclease ◽  
Gene Conversion Event ◽  
Group I Introns ◽  
Conversion Event ◽  
Homologous Recombination Event ◽  
Group I ◽  
Intron Homing

ABSTRACT Many group I introns encode endonucleases that promote intron homing by initiating a double-stranded break-mediated homologous recombination event. In this work we describe intron homing in Bacillus subtilis phages SPO1 and SP82. The introns encode the DNA endonucleases I-HmuI and I-HmuII, respectively, which belong to the H-N-H endonuclease family and possess nicking activity in vitro. Coinfections of B. subtilis with intron-minus and intron-plus phages indicate that I-HmuI and I-HmuII are required for homing of the SPO1 and SP82 introns, respectively. The homing process is a gene conversion event that does not require the major B. subtilis recombination pathways, suggesting that the necessary functions are provided by phage-encoded factors. Our results provide the first examples of H-N-H endonuclease-mediated intron homing and the first demonstration of intron homing initiated by a nicking endonuclease.

scholarly journals Rad51-Independent Interchromosomal Double-Strand Break Repair by Gene Conversion Requires Rad52 but Not Rad55, Rad57, or Dmc1

2007 ◽  
Vol 28 (3) ◽  
pp. 897-906 ◽  
Author(s):  
Thomas J. Pohl ◽  
Jac A. Nickoloff
Keyword(s):  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Single Strand ◽  
Homology Search ◽  
Wild Type ◽  
Dsb Repair ◽  
Single Strand Annealing ◽  
In The Wild ◽  
Break Induced Replication

ABSTRACT Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its “mediators,” including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Δ mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Δ mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Δ and rad52Δ mutants, but not in a rad51Δ rad52Δ double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Δ mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Δ mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.

scholarly journals Role of Exonucleolytic Degradation in Group I Intron Homing in Phage T4

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1501-1512 ◽  
Author(s):  
Yi-Jiun Huang ◽  
Monica M Parker ◽  
Marlene Belfort
Keyword(s):  
Insertion Site ◽  
Rnase H ◽  
Nucleic Acid Metabolism ◽  
Exonuclease Activity ◽  
Intron Insertion ◽  
Vitro Assay ◽  
Phage T4 ◽  
Group I ◽  
Intron Homing

AbstractHoming of the phage T4 td intron is initiated by the intron-encoded endonuclease I-TevI, which cleaves the intronless allele 23 and 25 nucleotides upstream of the intron insertion site (IS). The distance between the I-TevI cleavage site (CS) and IS implicates endo- and/or exonuclease activities to resect the DNA segment between the IS and CS. Furthermore, 3′ tails must presumably be generated for strand invasion by 5′-3′ exonuclease activity. Three experimental approaches were used to probe for phage nucleases involved in homing: a comparative analysis of in vivo homing levels of nuclease-deficient phage, an in vitro assay of nuclease activity and specificity, and a coconversion analysis of flanking exon markers. It was thereby demonstrated that T4 RNase H, a 5′-3′ exonuclease, T4 DNA exonuclease A (DexA) and the exonuclease activity of T4 DNA polymerase (43Exo), 3′-5′ exonucleases, play a role in intron homing. The absence of these functions impacts not only homing efficiency but also the extent of degradation and flanking marker coconversion. These results underscore the critical importance of the 3′ tail in intron homing, and they provide the first direct evidence of a role for 3′ single-stranded DNA ends as intermediates in T4 recombination. Also, the involvement of RNase H, DexA, and 43Exo in homing provides a clear example of the harnessing of functions variously involved in phage nucleic acid metabolism for intron propagation.

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