scholarly journals cDNA of the Yeast Retrotransposon Ty5 Preferentially Recombines with Substrates in Silent Chromatin

1999 ◽  
Vol 19 (1) ◽  
pp. 484-494 ◽  
Author(s):  
Ning Ke ◽  
Daniel F. Voytas
Keyword(s):  
Alternative Model ◽  
Point Mutations ◽  
Silent Chromatin ◽  
Chromosomal Locus ◽  
Homologous Sequences ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Telomere Dynamics ◽  
Recombination Assay ◽  
Yeast Retrotransposon

ABSTRACTThe yeast retrotransposon Ty5 preferentially integrates into regions of silent chromatin. Ty5 cDNA also recombines with homologous sequences, generating tandem elements or elements that have exchanged markers between cDNA and substrate. In this study, we demonstrate that Ty5 integration depends upon the conserved DD(35)E domain of integrase andcis-acting sequences at the end of the long terminal repeat (LTR) implicated in integrase binding. cDNA recombination requires Rad52p, which is responsible for homologous recombination. Interestingly, Ty5 cDNA recombines at least three times more frequently with substrates in silent chromatin than with a control substrate at an internal chromosomal locus. This preference depends upon the Ty5 targeting domain that is responsible for integration specificity, suggesting that localization of cDNA to silent chromatin results in the enhanced recombination. Recombination with a telomeric substrate occasionally generates highly reiterated Ty5 arrays, and mechanisms for tandem element formation were explored by using a plasmid-based recombination assay. Point mutations were introduced into plasmid targets, and recombination products were characterized to determine recombination initiation sites. Despite our previous observation of the importance of the LTR in forming tandem elements, recombination cannot simply be explained by crossover events between the LTRs of substrate and cDNA. We propose an alternative model based on single-strand annealing, where single-stranded cDNA initiates tandem element formation and the LTR is required for strand displacement to form a looped intermediate. Retrotransposons are increasingly found associated with chromosome ends, and amplification of Ty5 by both integration and recombination exemplifies how retroelements can contribute to telomere dynamics.

BCR-ABL Induces Error-Prone Single Strand Annealing in Transformed Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2937-2937
Author(s):  
Margret S. Rodrigues ◽  
Jeffrey R. Gonneville ◽  
Klaus Podar ◽  
David M. Weinstock ◽  
James D. Griffin ◽  
...  
Keyword(s):  
Stromal Cell ◽  
Kinase Activity ◽  
Point Mutations ◽  
Dna Lesions ◽  
Single Strand ◽  
Myelogenous Leukemia ◽  
Transformed Cells ◽  
Abl Kinase ◽  
Single Strand Annealing ◽  
Strand Annealing

Abstract Chronic myelogenous leukemia (CML) is a hematopoietic stem cell disorder, caused by the BCR-ABL tyrosine kinase oncogene. BCR-ABL kinase activity is required for all aspects of transformation, including abnormal proliferation and neoplastic expansion of stem cells, activation of signaling pathways and genomic instability. We have recently shown that BCR-ABL kinase activity also causes elevated intracellular levels of reactive oxygen species (ROS). These chronically elevated ROS levels have been implicated in the induction of DNA double-strand breaks (DSB) and therapy-related drug resistance, mainly through low- fidelity homology-directed repair (HDR) of DNA lesions. We have utilized GFP-based reporters in BCR-ABL transformed cells to measure both HDR and single-strand annealing (SSA), a mutagenic pathway of homologous repair between repetitive sequences. Repair rates of a single DSB by each of these pathways was measured in BCR-ABL expressing BaF3 cells (BaF3.p210), which have previously been shown to be an excellent system to model the induction of imatinib resistant mutations. We found that inhibition of BCR-ABL kinase activity by imatinib decreased the frequency of SSA by 73 ± 2% (n=3) and increased the frequency of HDR by 70 ± 11% (n=3) in BaF3.p210, compared to untreated cells. Treatment with imatinib did not affect HDR or SSA in BaF3 cells that do not express BCR-ABL. We also determined the fidelity of both SSA and HDR in our model system using clonal populations that stably express the repaired GFP+ reporters (n=20). As expected, sequencing of GFP+ repair products from cells containing the SSA reporter confirmed the expected sequence deletions, consistent with an error-prone mechanism. In contrast, sequencing of GFP+ repair products from the HDR reporter indicated a high-fidelity repair mechanism, without any mutations. It should be noted that this reporter assay does not account for potentially imprecise HDR, leading to a non-functional GFP. Nevertheless, point mutations at the previously reported rate were not detected. Thus, altered HDR fidelity may not be a universal mechanism for the induction of point mutations by BCR-ABL. Our data suggests a unique and alternative pathway, whereby BCR-ABL alters the balance between the SSA and HDR pathways. Moreover, in the presence of interleukin-3 at a concentration that supports not only viability but also cell growth (1ng/ml), imatinib was ineffective at protecting cells from error-prone SSA. In vivo, stromal cells may provide these additional growth signals. Our in vitro data show that stromal cell conditioned medium is not only sufficient to support growth and viability of CML cell lines in the presence of imatinib, but it can also lead to elevated levels of ROS. An abnormal increase in ROS is sufficient by itself to cause genomic mutations (transitions and/or transversions) as a result of single strand oxidative DNA lesions, even in the absence of altered DSB or single strand lesion repair mechanisms. Thus, therapy-related resistance and additional genomic abnormalities may not only be a result of BCR-ABL dependent ROS induction but may also occur within the stromal cell microenvironment through ROS induction by growth factors.

scholarly journals Characterization of RAD51-Independent Break-Induced Replication That Acts Preferentially with Short Homologous Sequences

2002 ◽  
Vol 22 (18) ◽  
pp. 6384-6392 ◽  
Author(s):  
Grzegorz Ira ◽  
James E. Haber
Keyword(s):  
Double Strand Breaks ◽  
Inverted Repeats ◽  
Strand Breaks ◽  
Homologous Sequences ◽  
Recombination Processes ◽  
Single Strand Annealing ◽  
Strand Invasion ◽  
Strand Annealing ◽  
Break Induced Replication

ABSTRACT Repair of double-strand breaks by gene conversions between homologous sequences located on different Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences between the RAD51- and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the absence of telomerase.

Yeastspt6-140Mutation, Affecting Chromatin and Transcription, Preferentially Increases Recombination in Which Rad51p-Mediated Strand Exchange Is Dispensable

Genetics ◽  
2001 ◽  
Vol 158 (2) ◽  
pp. 597-611 ◽  
Author(s):  
Francisco Malagón ◽  
Andrés Aguilera
Keyword(s):  
Strand Exchange ◽  
Micrococcal Nuclease ◽  
Sensitivity Analyses ◽  
Inverted Repeats ◽  
Major Mechanism ◽  
Spontaneous Recombination ◽  
Single Strand Annealing ◽  
Strand Invasion ◽  
Strand Annealing ◽  
Break Induced Replication

AbstractWe have shown that the spt6-140 and spt4-3 mutations, affecting chromatin structure and transcription, stimulate recombination between inverted repeats by a RAD52-dependent mechanism that is very efficient in the absence of RAD51, RAD54, RAD55, and RAD57. Such a mechanism of recombination is RAD1-RAD59-dependent and yields gene conversions highly associated with the inversion of the repeat. The spt6-140 mutation alters transcription and chromatin in our inverted repeats, as determined by Northern and micrococcal nuclease sensitivity analyses, respectively. Hyper-recombination levels are diminished in the absence of transcription. We believe that the chromatin alteration, together with transcription impairment caused by spt6-140, increases the incidence of spontaneous recombination regardless of whether or not it is mediated by Rad51p-dependent strand exchange. Our results suggest that spt6, as well as spt4, primarily stimulates a mechanism of break-induced replication. We discuss the possibility that the chromatin alteration caused by spt6-140 facilitates a Rad52p-mediated one-ended strand invasion event, possibly inefficient in wild-type chromatin. Our results are consistent with the idea that the major mechanism leading to inversions might not be crossing over but break-induced replication followed by single-strand annealing.

scholarly journals CHROMOSOMAL BASIS OF THE MEROZYGOSITY IN A PARTIALLY DIPLOID MUTANT OF PNEUMOCOCCUS

Genetics ◽  
1975 ◽  
Vol 80 (4) ◽  
pp. 667-678
Author(s):  
Mary Lee S Ledbetter ◽  
Rollin D Hotchkiss
Keyword(s):  
High Efficiency ◽  
Point Mutations ◽  
Resistant Mutant ◽  
Chromosomal Marker ◽  
Structural Abnormality ◽  
C Cells ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Low Efficiency ◽  
Derivatives Of

ABSTRACT A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

scholarly journals The Short Life Span of Saccharomyces cerevisiae sgs1 and srs2 Mutants Is a Composite of Normal Aging Processes and Mitotic Arrest Due to Defective Recombination

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1531-1542 ◽  
Author(s):  
Mitch McVey ◽  
Matt Kaeberlein ◽  
Heidi A Tissenbaum ◽  
Leonard Guarente
Keyword(s):  
Cell Cycle ◽  
Life Span ◽  
Mitotic Cell ◽  
Dna Helicase ◽  
Premature Aging ◽  
Growth Defect ◽  
Short Life Span ◽  
Single Strand Annealing ◽  
Late Generation ◽  
Strand Annealing

Abstract Evidence from many organisms indicates that the conserved RecQ helicases function in the maintenance of genomic stability. Mutation of SGS1 and WRN, which encode RecQ homologues in budding yeast and humans, respectively, results in phenotypes characteristic of premature aging. Mutation of SRS2, another DNA helicase, causes synthetic slow growth in an sgs1 background. In this work, we demonstrate that srs2 mutants have a shortened life span similar to sgs1 mutants. Further dissection of the sgs1 and srs2 survival curves reveals two distinct phenomena. A majority of sgs1 and srs2 cells stops dividing stochastically as large-budded cells. This mitotic cell cycle arrest is age independent and requires the RAD9-dependent DNA damage checkpoint. Late-generation sgs1 and srs2 cells senesce due to apparent premature aging, most likely involving the accumulation of extrachromosomal rDNA circles. Double sgs1 srs2 mutants are viable but have a high stochastic rate of terminal G2/M arrest. This arrest can be suppressed by mutations in RAD51, RAD52, and RAD57, suggesting that the cell cycle defect in sgs1 srs2 mutants results from inappropriate homologous recombination. Finally, mutation of RAD1 or RAD50 exacerbates the growth defect of sgs1 srs2 cells, indicating that sgs1 srs2 mutants may utilize single-strand annealing as an alternative repair pathway.

scholarly journals The Yeast Recombinational Repair Protein Rad59 Interacts With Rad52 and Stimulates Single-Strand Annealing

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 515-525 ◽  
Author(s):  
Allison P Davis ◽  
Lorraine S Symington
Keyword(s):  
Protein A ◽  
Single Strand ◽  
Physical Interaction ◽  
Dna Double Strand Breaks ◽  
Single Stranded Dna ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Recombination Pathway

Abstract The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.

scholarly journals Saw1 localizes to repair sites but is not required for recruitment of Rad10 to repair intermediates bearing short non-homologous 3′ flaps during single-strand annealing in S. cerevisiae

2015 ◽  
Vol 412 (1-2) ◽  
pp. 131-139
Author(s):  
Melina Mardirosian ◽  
Linette Nalbandyan ◽  
Aaron D. Miller ◽  
Claire Phan ◽  
Eric P. Kelson ◽  
...  
Keyword(s):  
Single Strand ◽  
Single Strand Annealing ◽  
Strand Annealing

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells

1994 ◽  
Vol 14 (1) ◽  
pp. 400-406
Author(s):  
W P Deng ◽  
J A Nickoloff
Keyword(s):  
Dna Replication ◽  
Mismatch Repair ◽  
Mammalian Cells ◽  
Strand Break ◽  
Wild Type ◽  
Heteroduplex Dna ◽  
Frameshift Mutations ◽  
Single Strand Annealing ◽  
Extrachromosomal Recombination ◽  
Strand Annealing

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.

scholarly journals Rejoining of DNA Double-Strand Breaks as a Function of Overhang Length

2005 ◽  
Vol 25 (3) ◽  
pp. 896-906 ◽  
Author(s):  
James M. Daley ◽  
Thomas E. Wilson
Keyword(s):  
Gc Content ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Primary Determinant ◽  
Overhang Length ◽  
Strand Annealing

ABSTRACT The ends of spontaneously occurring double-strand breaks (DSBs) may contain various lengths of single-stranded DNA, blocking lesions, and gaps and flaps generated by end annealing. To investigate the processing of such structures, we developed an assay in which annealed oligonucleotides are ligated onto the ends of a linearized plasmid which is then transformed into Saccharomyces cerevisiae. Reconstitution of a marker occurs only when the oligonucleotides are incorporated and repair is in frame, permitting rapid analysis of complex DSB ends. Here, we created DSBs with compatible overhangs of various lengths and asked which pathways are required for their precise repair. Three mechanisms of rejoining were observed, regardless of overhang polarity: nonhomologous end joining (NHEJ), a Rad52-dependent single-strand annealing-like pathway, and a third mechanism independent of the first two mechanisms. DSBs with overhangs of less than 4 bases were mainly repaired by NHEJ. Repair became less dependent on NHEJ when the overhangs were longer or had a higher GC content. Repair of overhangs greater than 8 nucleotides was as much as 150-fold more efficient, impaired 10-fold by rad52 mutation, and highly accurate. Reducing the microhomology extent between long overhangs reduced their repair dramatically, to less than NHEJ of comparable short overhangs. These data support a model in which annealing energy is a primary determinant of the rejoining efficiency and mechanism.

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