Gene conversion in the chicken immunoglobulin locus: A paradigm of homologous recombination in higher eukaryotes

1994 ◽  
Vol 50 (3) ◽  
pp. 270-276 ◽  
Author(s):  
O. Y. Bezzubova ◽  
J. -M. Buerstedde
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Immunoglobulin Locus ◽  
Higher Eukaryotes

scholarly journals The 9-1-1 DNA Clamp Is Required for Immunoglobulin Gene Conversion

2008 ◽  
Vol 28 (19) ◽  
pp. 6113-6122 ◽  
Author(s):  
Alihossein Saberi ◽  
Makoto Nakahara ◽  
Julian E. Sale ◽  
Koji Kikuchi ◽  
Hiroshi Arakawa ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Translesion Synthesis ◽  
Strand Break ◽  
Double Strand Break ◽  
Immunoglobulin Gene ◽  
Abasic Sites ◽  
Immunoglobulin Locus ◽  
Activation Induced Deaminase ◽  
Dt40 Cells

ABSTRACT Chicken DT40 cells deficient in the 9-1-1 checkpoint clamp exhibit hypersensitivity to a variety of DNA-damaging agents. Although recent work suggests that, in addition to its role in checkpoint activation, this complex may play a role in homologous recombination and translesion synthesis, the cause of this hypersensitivity has not been studied thoroughly. The immunoglobulin locus of DT40 cells allows monitoring of homologous recombination and translesion synthesis initiated by activation-induced deaminase (AID)-dependent abasic sites. We show that both the RAD9 −/− and RAD17 −/− mutants exhibit substantially reduced immunoglobulin gene conversion. However, the level of nontemplated immunoglobulin point mutation increased in these mutants, a finding that is reminiscent of the phenotype resulting from the loss of RAD51 paralogs or Brca2. This suggests that the 9-1-1 complex does not play a central role in translesion synthesis in this context. Despite reduced immunoglobulin gene conversion, the RAD9 −/− and RAD17 −/− cells do not exhibit a prominent defect in double-strand break-induced gene conversion or a sensitivity to camptothecin. This suggests that the roles of Rad9 and Rad17 may be confined to a subset of homologous recombination reactions initiated by replication-stalling lesions rather than those associated with double-strand break repair.

Gene replacement with one-sided homologous recombination

1992 ◽  
Vol 12 (1) ◽  
pp. 360-367
Author(s):  
N Berinstein ◽  
N Pennell ◽  
C A Ottaway ◽  
M J Shulman
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Target Gene ◽  
Gene Replacement ◽  
Immunoglobulin Genes ◽  
Nonhomologous Recombination ◽  
Immunoglobulin Locus ◽  
Chromosomal Sequences ◽  
Dna Vectors

Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.

scholarly journals Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks

2009 ◽  
Vol 29 (17) ◽  
pp. 4742-4756 ◽  
Author(s):  
Alexander Lorenz ◽  
Fekret Osman ◽  
Victoria Folkyte ◽  
Sevil Sofueva ◽  
Matthew C. Whitby
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Replication Fork ◽  
Direct Repeat ◽  
Dna Helicase ◽  
Replication Forks ◽  
Site Specific ◽  
Replicative Stress ◽  
Recombinant Formation ◽  
A Site

ABSTRACT Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.

scholarly journals Genetic Requirements for RAD51- andRAD54-Independent Break-Induced Replication Repair of a Chromosomal Double-Strand Break

2001 ◽  
Vol 21 (6) ◽  
pp. 2048-2056 ◽  
Author(s):  
Laurence Signon ◽  
Anna Malkova ◽  
Maria L. Naylor ◽  
Hannah Klein ◽  
James E. Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Telomere Maintenance ◽  
Diploid Cells ◽  
Break Induced Replication ◽  
In Cells

ABSTRACT Broken chromosomes can be repaired by several homologous recombination mechanisms, including gene conversion and break-induced replication (BIR). In Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) is normally repaired by gene conversion. Previously, we have shown that in the absence ofRAD52, repair is nearly absent and diploid cells lose the broken chromosome; however, in cells lacking RAD51, gene conversion is absent but cells can repair the DSB by BIR. We now report that gene conversion is also abolished when RAD54, RAD55, and RAD57 are deleted but BIR occurs, as withrad51Δ cells. DSB-induced gene conversion is not significantly affected when RAD50, RAD59, TID1(RDH54), SRS2, or SGS1 is deleted. Various double mutations largely eliminate both gene conversion and BIR, including rad51Δ rad50Δ, rad51Δ rad59Δ, andrad54Δ tid1Δ. These results demonstrate that there is aRAD51- and RAD54-independent BIR pathway that requires RAD59, TID1, RAD50, and presumablyMRE11 and XRS2. The similar genetic requirements for BIR and telomere maintenance in the absence of telomerase also suggest that these two processes proceed by similar mechanisms.

scholarly journals Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

1997 ◽  
Vol 17 (11) ◽  
pp. 6386-6393 ◽  
Author(s):  
D G Taghian ◽  
J A Nickoloff
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Chromosomal Dna ◽  
Exogenous Dna ◽  
Strand Breaks ◽  
Conversion Tract

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

scholarly journals Duplication of chickendefensin7gene generated by gene conversion and homologous recombination

2016 ◽  
Vol 113 (48) ◽  
pp. 13815-13820 ◽  
Author(s):  
Mi Ok Lee ◽  
Susanne Bornelöv ◽  
Leif Andersson ◽  
Susan J. Lamont ◽  
Junfeng Chen ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Copy Number Variation ◽  
Gene Conversion ◽  
Copy Number ◽  
Computational Prediction ◽  
Gene Families ◽  
Duplication Event ◽  
Promoter Regions ◽  
Large Gene ◽  
Number Variation

Defensins constitute an evolutionary conserved family of cationic antimicrobial peptides that play a key role in host innate immune responses to infection. Defensin genes generally reside in complex genomic regions that are prone to structural variation, and defensin genes exhibit extensive copy number variation in humans and in other species. Copy number variation of defensin genes was examined in inbred lines of Leghorn and Fayoumi chickens, and a duplication ofdefensin7was discovered in the Fayoumi breed. Analysis of junction sequences confirmed the occurrence of a simple tandem duplication ofdefensin7with sequence identity at the junction, suggesting nonallelic homologous recombination betweendefensin7anddefensin6. The duplication event generated two chimeric promoters that are best explained by gene conversion followed by homologous recombination. Expression ofdefensin7was not elevated in animals with two genes despite both genes being transcribed in the tissues examined. Computational prediction of promoter regions revealed the presence of several putative transcription factor binding sites generated by the duplication event. These data provide insight into the evolution and possible function of large gene families and specifically, the defensins.

scholarly journals Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (7) ◽  
pp. 2329-2334 ◽  
Author(s):  
J R Simon ◽  
P D Moore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Mutant Gene ◽  
Transformation Process ◽  
Single Stranded Dna ◽  
Autonomous Replication ◽  
Double Stranded Dna ◽  
Chromosomal Genes ◽  
Chromosomal Sequences

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.

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