scholarly journals Biased Gene Conversion in Rhizobium etli Is Caused by Preferential Double-Strand Breaks on One of the Recombining Homologs

2015 ◽  
Vol 198 (3) ◽  
pp. 591-599 ◽  
Author(s):  
Fares Osam Yáñez-Cuna ◽  
Mildred Castellanos ◽  
David Romero
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Biased Gene Conversion ◽  
Resident Plasmid

ABSTRACTGene conversion, the nonreciprocal transfer of information during homologous recombination, is the main process that maintains identity between members of multigene families. Gene conversion in the nitrogenase (nifH) multigene family ofRhizobium etliwas analyzed by using a two-plasmid system, where each plasmid carried a copy ofnifH. One of thenifHcopies was modified, creating restriction fragment length polymorphisms (RFLPs) spaced along the gene. Once the modified plasmid was introduced intoR. etli, selection was done for cointegration with a resident plasmid lacking the RFLPs. Most of the cointegrate molecules harbor gene conversion events, biased toward a gain of RFLPs. This bias may be explained under the double-strand break repair model by proposing that thenifHgene lacking the RFLPs suffers a DNA double-strand break, so the incoming plasmid functions as a template for repairing the homolog on the resident plasmid. To support this proposal, we cloned an SceI site into thenifHhomolog that had the RFLPs used for scoring gene conversion.In vivoexpression of the meganuclease I-SceI allowed the generation of a double-strand break on this homolog. Upon introduction of this modified plasmid into anR. etlistrain lacking I-SceI, biased gene conversion still favored the retention of markers on the incoming plasmid. In contrast, when the recipient strain ectopically expressed I-SceI, a dramatic reversal in gene conversion bias was seen, favoring the preservation of resident sequences. These results show that biased gene conversion is caused by preferential double-strand breaks on one of the recombining homologs.IMPORTANCEIn this work, we analyzed gene conversion by using a system that entails horizontal gene transfer followed by homologous recombination in the recipient cell. Most gene conversion events are biased toward the acquisition of the incoming sequences, ranging in size from 120 bp to 800 bp. This bias is due to preferential cutting of resident DNA and can be reversed upon introduction of a double-strand break on the incoming sequence. Since conditions used in this work are similar to those in horizontal gene transfer, it provides evidence that, upon transfer, the resident DNA preferentially acquires gene variants.

scholarly journals Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair

2020 ◽  
Vol 48 (15) ◽  
pp. 8490-8508 ◽  
Author(s):  
Sarah S Henrikus ◽  
Camille Henry ◽  
Amy E McGrath ◽  
Slobodan Jergic ◽  
John P McDonald ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Single Molecule ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
E Coli ◽  
Pol Iv ◽  
Dna Polymerase Iv

Abstract Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

An Immortalised Leukaemia Cell Line Models the Quiescence of Leukaemic Stem Cells and Shows Impaired Double Strand Break Repair Following Daunorubicin Treatment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3989-3989
Author(s):  
Claire Seedhouse ◽  
Abigail Whittall ◽  
Karuna Tandon ◽  
Nigel H. Russell ◽  
Monica Pallis
Keyword(s):  
Cell Cycle ◽  
Strand Break ◽  
Multidrug Resistant ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Proliferating Cells ◽  
Drug Induced ◽  
Strand Breaks ◽  
Damage Response ◽  
Mitochondrial Pore

Abstract Abstract 3989 Objective: Approximately 50% of patients with acute myeloid leukaemia respond to remission-induction chemotherapy, but later relapse. Relapse is thought to be due to the continued presence of a quiescent, chemoresistant leukaemic cell subpopulation. Understanding the damage response in these cells might help to guide targeted therapies. We therefore developed an in vitro model of the quiescent subpopulation and used it to study drug-induced damage and repair in quiescent multidrug resistant cells. Methods: We cultured CD34+ CD38- multidrug resistant KG1a AML cells under several conditions reported to induce cell cycle arrest. We used Pyronin Y to measure RNA content and 7-aminoactinomycin D to measure cell viability. Chemosensitivity, reactive oxygen species (ROS), mitochondrial pore transition and oxidative damage were measured flow cytometrically. gammaH2A.X foci were quantified to measure the double strand break response and DNA damage response and repair gene expression was studied using PCR microarrays and confirmed by real time PCR. Results: mTOR inhibitors induced an increase in G0 without induction of apoptosis. 48 hours' exposure to rapamycin increased the proportion of G0 cells from 13.3% (SD 2.3%) to 46.1% (SD 6%) and decreased mean cell volume. Delayed re-entry into cell cycle following rapamycin withdrawal confirmed the G0 status of these cells. Differentiation markers remained negative. Although several of the other conditions studied resulted in reduced cell growth, they also induced apoptosis, as did combinations of rapamycin with other growth inhibitors. The toxicity of the chemotherapy drug daunorubicin, which acts in part by inducing ROS, was reduced in the quiescence-enriched cells. Sensitivity to mitochondrial pore transition was similar in proliferating and quiescence-enriched cells, indicating that apoptotic pathways are not impaired. However, both basal and drug-induced ROS were significantly lower in quiescence-enriched than in the proliferating cells (p=0.006 for basal ROS and 0.013 for daunorubicin-induced ROS). Furthermore, several DNA repair genes were differentially regulated following daunorubicin treatment of the quiescence-enriched compared to the proliferating cells – these included genes responsible for the repair of double strand breaks. On treatment with daunorubicin, double strand breaks, but not oxidative damage to DNA were observed in both cell populations. However, strikingly, although quiescence-enriched cells sustained fewer DNA damage foci than proliferating cells, they were unable to resolve the damage after daunorubicin was removed. Conclusion: By using rapamycin to enrich KG1a cells for quiescence, we have shown low basal and drug-induced ROS to be associated with chemoresistance in these cells. However, we also found that quiescence gave rise to an impaired double strand break response, which might force these cells to rely on alternative repair pathways and thus be sensitive to synthetic lethal targeting. Disclosures: No relevant conflicts of interest to declare.

scholarly journals SIRT6 is a DNA Double-Strand Break Sensor

2019 ◽  
Author(s):  
Lior Onn ◽  
Miguel Portillo ◽  
Stefan Ilic ◽  
Gal Cleitman ◽  
Daniel Stein ◽  
...  
Keyword(s):  
Dna Damage ◽  
Binding Capacity ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
The Road ◽  
Non Homologous End Joining

AbstractDNA double strand breaks are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure, with high affinity for double strand breaks. It relocates to sites of damage independently of signalling and known sensors and activates downstream signalling cascades for double strand break repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the Homologous Recombination and Non-Homologous End Joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as DNA damage sensor, which is critical for initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB binding capacity and DDR activation. SIRT6 activates the DDR, before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 at the top of the DDR and pave the road to dissect the contributions of distinct double strand break sensors in downstream signalling.

scholarly journals Exosome-mediated horizontal gene transfer occurs in double-strand break repair during genome editing

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Ryuichi Ono ◽  
Yukuto Yasuhiko ◽  
Ken-ichi Aisaki ◽  
Satoshi Kitajima ◽  
Jun Kanno ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Genome Editing ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Break Repair

scholarly journals Repair of DNA Double-Strand Breaks following UV Damage in Three Sulfolobussolfataricus Strains

2010 ◽  
Vol 192 (19) ◽  
pp. 4954-4962 ◽  
Author(s):  
Michael L. Rolfsmeier ◽  
Marian F. Laughery ◽  
Cynthia A. Haseltine
Keyword(s):  
Sulfolobus Solfataricus ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Hyperthermophilic Archaea ◽  
Chromosomal Damage ◽  
Strand Breaks ◽  
Break Repair ◽  
Uv C

ABSTRACT DNA damage repair mechanisms have been most thoroughly explored in the eubacterial and eukaryotic branches of life. The methods by which members of the archaeal branch repair DNA are significantly less well understood but have been gaining increasing attention. In particular, the approaches employed by hyperthermophilic archaea have been a general source of interest, since these organisms thrive under conditions that likely lead to constant chromosomal damage. In this work we have characterized the responses of three Sulfolobus solfataricus strains to UV-C irradiation, which often results in double-strand break formation. We examined S. solfataricus strain P2 obtained from two different sources and S. solfataricus strain 98/2, a popular strain for site-directed mutation by homologous recombination. Cellular recovery, as determined by survival curves and the ability to return to growth after irradiation, was found to be strain specific and differed depending on the dose applied. Chromosomal damage was directly visualized using pulsed-field gel electrophoresis and demonstrated repair rate variations among the strains following UV-C irradiation-induced double-strand breaks. Several genes involved in double-strand break repair were found to be significantly upregulated after UV-C irradiation. Transcript abundance levels and temporal expression patterns for double-strand break repair genes were also distinct for each strain, indicating that these Sulfolobus solfataricus strains have differential responses to UV-C-induced DNA double-strand break damage.

Regulation of the cellular DNA double-strand break response

2007 ◽  
Vol 85 (6) ◽  
pp. 663-674 ◽  
Author(s):  
Kendra L. Cann ◽  
Geoffrey G. Hicks
Keyword(s):  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Cell Cycle Checkpoints ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Cellular Dna

DNA double-strand breaks occur frequently in cycling cells, and are also induced by exogenous sources, including ionizing radiation. Cells have developed integrated double-strand break response pathways to cope with these lesions, including pathways that initiate DNA repair (either via homologous recombination or nonhomologous end joining), the cell-cycle checkpoints (G1–S, intra-S phase, and G2–M) that provide time for repair, and apoptosis. However, before any of these pathways can be activated, the damage must first be recognized. In this review, we will discuss how the response of mammalian cells to DNA double-strand breaks is regulated, beginning with the activation of ATM, the pinnacle kinase of the double-strand break signalling cascade.

scholarly journals Mutations in the Saccharomyces cerevisiae CDC1 gene affect double-strand-break-induced intrachromosomal recombination.

1994 ◽  
Vol 14 (12) ◽  
pp. 8037-8050 ◽  
Author(s):  
J Halbrook ◽  
M F Hoekstra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Essential Gene ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Intrachromosomal Recombination ◽  
Spontaneous Frequency ◽  
Type Strains

To isolate Saccharomyces cerevisiae mutants defective in recombinational DNA repair, we constructed a strain that contains duplicated ura3 alleles that flank LEU2 and ADE5 genes at the ura3 locus on chromosome V. When a HO endonuclease cleavage site is located within one of the ura3 alleles, Ura+ recombination is increased over 100-fold in wild-type strains following HO induction from the GAL1, 10 promoter. This strain was used to screen for mutants that exhibited reduced levels of HO-induced intrachromosomal recombination without significantly affecting the spontaneous frequency of Ura+ recombination. One of the mutations isolated through this screen was found to affect the essential gene CDC1. This mutation, cdc1-100, completely eliminated HO-induced Ura+ recombination yet maintained both spontaneous preinduced recombination levels and cell viability, cdc1-100 mutants were moderately sensitive to killing by methyl methanesulfonate and gamma irradiation. The effect of the cdc1-100 mutation on recombinational double-strand break repair indicates that a recombinationally silent mechanism other than sister chromatid exchange was responsible for the efficient repair of DNA double-strand breaks.

scholarly journals YGR042W/MTE1 Functions in Double-Strand Break Repair with MPH1

2015 ◽  
Author(s):  
Askar Yimit ◽  
TaeHyung Kim ◽  
Ranjith Anand ◽  
Sarah Meister ◽  
Jiongwen Ou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Strand Break ◽  
Double Strand Break ◽  
Dna Helicase ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Dependent Manner ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Break Repair

Double-strand DNA breaks occur upon exposure of cells to agents such as ionizing radiation and ultraviolet light or indirectly through replication fork collapse at DNA damage sites. If left unrepaired double-strand breaks can cause genome instability and cell death. In response to DNA damage, proteins involved in double-strand break repair by homologous recombination re-localize into discrete nuclear foci. We identified 29 proteins that co-localize with the recombination repair protein Rad52 in response to DNA damage. Of particular interest, Ygr042w/Mte1, a protein of unknown function, showed robust colocalization with Rad52. Mte1 foci fail to form when the DNA helicase Mph1 is absent. Mte1 and Mph1 form a complex, and are recruited to double-strand breaks in vivo in a mutually dependent manner. Mte1 is important for resolution of Rad52 foci during double-strand break repair, and for suppressing break-induced replication. Together our data indicate that Mte1 functions with Mph1 in double-strand break repair.

scholarly journals In Vitro Repair of Gaps in Bacteriophage T7 DNA

1998 ◽  
Vol 180 (23) ◽  
pp. 6193-6202 ◽  
Author(s):  
Ying-Ta Lai ◽  
Warren Masker
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Bacteriophage T7 ◽  
Dna Molecules ◽  
Strand Breaks ◽  
Highly Efficient ◽  
Break Repair

ABSTRACT An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.

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