scholarly journals Focused Genetic Recombination of Bacteriophage T4 Initiated by Double-Strand Breaks

Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 543-556
Author(s):  
Victor Shcherbakov ◽  
Igor Granovsky ◽  
Lidiya Plugina ◽  
Tamara Shcherbakova ◽  
Svetlana Sizova ◽  
...  
Keyword(s):  
Genetic Recombination ◽  
Bacteriophage T4 ◽  
Proximal Part ◽  
Coupling Model ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
The Right ◽  
Frequency Distance

Abstract A model system for studying double-strand-break (DSB)-induced genetic recombination in vivo based on the ets1 segCΔ strain of bacteriophage T4 was developed. The ets1, a 66-bp DNA fragment of phage T2L containing the cleavage site for the T4 SegC site-specific endonuclease, was inserted into the proximal part of the T4 rIIB gene. Under segC+ conditions, the ets1 behaves as a recombination hotspot. Crosses of the ets1 against rII markers located to the left and to the right of ets1 gave similar results, thus demonstrating the equal and symmetrical initiation of recombination by either part of the broken chromosome. Frequency/distance relationships were studied in a series of two- and three-factor crosses with other rIIB and rIIA mutants (all segC+) separated from ets1 by 12-2100 bp. The observed relationships were readily interpretable in terms of the modified splice/patch coupling model. The advantages of this localized or focused recombination over that distributed along the chromosome, as a model for studying the recombination-replication pathway in T4 in vivo, are discussed.

scholarly journals βarrestin-1 regulates DNA repair by acting as an E3-ubiquitin ligase adaptor for 53BP1

2019 ◽  
Vol 27 (4) ◽  
pp. 1200-1213 ◽  
Author(s):  
Ainhoa Nieto ◽  
Makoto R. Hara ◽  
Victor Quereda ◽  
Wayne Grant ◽  
Vanessa Saunders ◽  
...  
Keyword(s):  
Dna Damage ◽  
Ionizing Radiation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Molecular Scaffold ◽  
Strand Breaks

Abstract Cellular DNA is constantly under threat from internal and external insults, consequently multiple pathways have evolved to maintain chromosomal fidelity. Our previous studies revealed that chronic stress, mediated by continuous stimulation of the β2-adrenergic-βarrestin-1 signaling axis suppresses activity of the tumor suppressor p53 and impairs genomic integrity. In this pathway, βarrestin-1 (βarr1) acts as a molecular scaffold to promote the binding and degradation of p53 by the E3-ubiquitin ligase, MDM2. We sought to determine whether βarr1 plays additional roles in the repair of DNA damage. Here we demonstrate that in mice βarr1 interacts with p53-binding protein 1 (53BP1) with major consequences for the repair of DNA double-strand breaks. 53BP1 is a principle component of the DNA damage response, and when recruited to the site of double-strand breaks in DNA, 53BP1 plays an important role coordinating repair of these toxic lesions. Here, we report that βarr1 directs 53BP1 degradation by acting as a scaffold for the E3-ubiquitin ligase Rad18. Consequently, knockdown of βarr1 stabilizes 53BP1 augmenting the number of 53BP1 DNA damage repair foci following exposure to ionizing radiation. Accordingly, βarr1 loss leads to a marked increase in irradiation resistance both in cells and in vivo. Thus, βarr1 is an important regulator of double strand break repair, and disruption of the βarr1/53BP1 interaction offers an attractive strategy to protect cells against high levels of exposure to ionizing radiation.

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.

scholarly journals Loss of YY1 Impacts the Heterochromatic State and Meiotic Double-Strand Breaks during Mouse Spermatogenesis

2009 ◽  
Vol 29 (23) ◽  
pp. 6245-6256 ◽  
Author(s):  
Su Wu ◽  
Yueh-Chiang Hu ◽  
Huifei Liu ◽  
Yang Shi
Keyword(s):  
Structural Integrity ◽  
Critical Role ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Model Organisms ◽  
Global Changes ◽  
Strand Breaks ◽  
Yin Yang 1 ◽  
Insight Into

ABSTRACT The progression of spermatogenesis involves global changes in chromatin structure and conformation. However, our understanding of the regulation of chromatin changes in germ cells remains limited. Here we describe both in vivo RNA interference and genetic mouse knockout studies that identify a critical role for Yin Yang 1 (YY1) in mammalian spermatogenesis. In the YY1-deficient spermatocytes, we find a significant decrease in the global level of the heterochromatin markers (H3K9me3 and HP1-gamma) and a concomitant increase in the double-strand break (DSB) signals on chromosomes (gamma-H2AX, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, and Rad51) at the leptotene/zygotene stages of spermatocytes. These findings support a link between chromatin modifications and meiotic DSB formation, as has been seen in other model organisms. We propose that a depletion of YY1 may alter the structural integrity of heterochromatin, rendering it more accessible to the DSB machinery. In addition, YY1-deficient spermatocytes show univalent formation, increased aneuploidy, and pachytene cell death, which are likely due to defects in DNA repair. Taken together, this study identifies an important role for YY1 in mouse meiosis and provides new insight into mechanisms that regulate mammalian spermatogenesis.

scholarly journals Control of meiotic double-strand-break formation by ATM: local and global views

2017 ◽  
Author(s):  
Agnieszka Lukaszewicz ◽  
Julian Lange ◽  
Scott Keeney ◽  
Maria Jasin
Keyword(s):  
Strand Break ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Future Research ◽  
Dna Double Strand Breaks ◽  
Time Loss ◽  
Strand Breaks ◽  
Research Perspectives ◽  
The Right ◽  
Break Formation

ABSTRACTDNA double-strand breaks (DSBs) generated by the SPO11 protein initiate meiotic recombination, an essential process for successful chromosome segregation during gametogenesis. The activity of SPO11 is controlled by multiple factors and regulatory mechanisms, such that the number of DSBs is limited and DSBs form at distinct positions in the genome and at the right time. Loss of this control can affect genome integrity or cause meiotic arrest by mechanisms that are not fully understood. Here we focus on the DSB-responsive kinase ATM and its functions in regulating meiotic DSB numbers and distribution. We review the recently discovered roles of ATM in this context, discuss their evolutionary conservation, and examine future research perspectives.

scholarly journals Mating type-like conversion promoted by the 2 micrograms circle site-specific recombinase: implications for the double-strand-gap repair model.

1986 ◽  
Vol 6 (11) ◽  
pp. 3831-3837 ◽  
Author(s):  
M Jayaram
Keyword(s):  
Mating Type ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Site Specific ◽  
Strand Breaks ◽  
Conversion Event ◽  
Yeast Mating ◽  
Mating Type Switching

Double-strand breaks in DNA are known to promote recombination in Saccharomyces cerevisiae. Yeast mating type switching, which is a highly efficient gene conversion event, is apparently initiated by a site-specific double-strand break. The 2 micrograms circle site-specific recombinase, FLP, has been shown to make double-strand breaks in its substrate DNA. By using a hybrid 2 micrograms circle::Tn5 plasmid, a portion of which resembles, in its DNA organization, the active (MAT) and the silent (HML) yeast mating type loci, it is shown that FLP mediates a conversion event analogous to mating type switching. Whereas the FLP site-specific recombination is not dependent on the RAD52 gene product, the FLP-induced conversion is abolished in a rad52 background. The FLP-promoted conversion in vivo can be faithfully reproduced by making a double-stranded gap in vitro in the vicinity of the FLP site and allowing the gap to be repaired in vivo.

scholarly journals Visualization of Repair of Double-Strand Breaks in the Bacteriophage T7 Genome without Normal DNA Replication

2000 ◽  
Vol 182 (2) ◽  
pp. 327-336 ◽  
Author(s):  
Ying-Ta Lai ◽  
Warren Masker
Keyword(s):  
Dna Synthesis ◽  
High Efficiency ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Bacteriophage T7 ◽  
Growth Step ◽  
Strand Breaks

ABSTRACT An in vitro system based on extracts of Escherichia coli infected with bacteriophage T7 is able to repair double-strand breaks in a T7 genome with efficiencies of 20% or more. To achieve this high repair efficiency it is necessary that the reaction mixtures contain molecules of donor DNA that bracket the double-strand break. Gaps as long as 1,600 nucleotides are repaired almost as efficiently as simple double-strand breaks. DNA synthesis was measured while repair was taking place. It was found that the amount of DNA synthesis associated with repair of a double-strand break was below the level of detection possible with this system. Furthermore, repair efficiencies were the same with or without normal levels of T7 DNA polymerase. However, the repair required the 5′→3′ exonuclease encoded by T7 gene 6. The high efficiency of DNA repair allowed visualization of the repaired product after in vitro repair, thereby assuring that the repair took place in vitro rather than during an in vivo growth step after packaging.

scholarly journals Annealing vs. Invasion in Phage λ Recombination

Genetics ◽  
1997 ◽  
Vol 147 (3) ◽  
pp. 961-977 ◽  
Author(s):  
Mary M Stahl ◽  
Lynn Thomason ◽  
Anthony R Poteete ◽  
Trudee Tarkowski ◽  
Andrei Kuzminov ◽  
...  
Keyword(s):  
Genetic Recombination ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Maximal Stimulation ◽  
Strand Invasion ◽  
Strand Annealing ◽  
Dna Ends ◽  
Mutant Cells ◽  
Reca Mutant

Genetic recombination catalyzed by λ's Red pathway was studied in rec  + and recA mutant bacteria by examining both intracellular λ DNA and mature progeny particles. Recombination of nonreplicating phage chromosomes was induced by double-strand breaks delivered at unique sites in vivo. In rec  + cells, cutting only one chromosome gave nearly maximal stimulation of recombination; the recombinants formed contained relatively short hybrid regions, suggesting strand invasion. In contrast, in recA mutant cells, cutting the two parental chromosomes at non-allelic sites was required for maximal stimulation; the recombinants formed tended to be hybrid over the entire region between the two cuts, implying strand annealing. We conclude that, in the absence of RecA and the presence of non-allelic DNA ends, the Red pathway of λ catalyzes recombination primarily by annealing.

scholarly journals Double-Strand Break Formation by the RAG Complex at the Bcl-2 Major Breakpoint Region and at Other Non-B DNA Structures In Vitro

2005 ◽  
Vol 25 (14) ◽  
pp. 5904-5919 ◽  
Author(s):  
Sathees C. Raghavan ◽  
Patrick C. Swanson ◽  
Yunmei Ma ◽  
Michael R. Lieber
Keyword(s):  
Chromosomal Translocation ◽  
Dna Structure ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Breakpoint Region ◽  
Strand Breaks ◽  
Dna Structures ◽  
Follicular Lymphomas

ABSTRACT The most common chromosomal translocation in cancer, t(14;18) at the 150-bp bcl-2 major breakpoint region (Mbr), occurs in follicular lymphomas. The bcl-2 Mbr assumes a non-B DNA conformation, thus explaining its distinctive fragility. This non-B DNA structure is a target of the RAG complex in vivo, but not because of its primary sequence. Here we report that the RAG complex generates at least two independent nicks that lead to double-strand breaks in vitro, and this requires the non-B DNA structure at the bcl-2 Mbr. A 3-bp mutation is capable of abolishing the non-B structure formation and the double-strand breaks. The observations on the bcl-2 Mbr reflect more general properties of the RAG complex, which can bind and nick at duplex-single-strand transitions of other non-B DNA structures, resulting in double-strand breaks in vitro. Hence, the present study reveals novel insight into a third mechanism of action of RAGs on DNA, besides the standard heptamer/nonamer-mediated cleavage in V(D)J recombination and the in vitro transposase activity.

scholarly journals Repair of Double-Strand Breaks in Bacteriophage T4 by a Mechanism That Involves Extensive DNA Replication

Genetics ◽  
1996 ◽  
Vol 143 (4) ◽  
pp. 1507-1520 ◽  
Author(s):  
James W George ◽  
Kenneth N Kreuzer
Keyword(s):  
Dna Replication ◽  
Bacteriophage T4 ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Holliday Junction ◽  
Plasmid Replication ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Endonuclease Vii ◽  
Genomic Recombination

Abstract We investigated double-strand break (dsb) repair in bacteriophage T4 using a physical assay that involves a plasmid substrate with two inverted DNA segments. A dsb introduced into one repeat during a T4 infection induces efficient dsb repair using the second repeat as a template. This reaction is characterized by the following interesting features. First, the dsb induces a repair reaction that is directly coupled to extensive plasmid replication; the repaired/replicated product is in the form of long plasmid concatemers. Second, repair of the dsb site is frequently associated with exchange of flanking DNA. Third, the repair reaction is absolutely dependent on the products of genes uvsX, uusY, 32, 46, and 59, which are also required for phage genomic recombination-dependent DNA replication. Fourth, the coupled repair/replication reaction is only partly dependent on endonuclease VII (gp49), suggesting that either another Holliday-junction-cleaving activity or an alternate resolution pathway is active during T4 infections. Because this repair reaction is directly coupled to extensive replication, it cannot be explained by the Szostak  et al. model. We present and discuss a model for the coupled repair/replication reaction, called the extensive chromosome replication model for dsb repair.

Mating type-like conversion promoted by the 2 micrograms circle site-specific recombinase: implications for the double-strand-gap repair model

1986 ◽  
Vol 6 (11) ◽  
pp. 3831-3837
Author(s):  
M Jayaram
Keyword(s):  
Mating Type ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Site Specific ◽  
Strand Breaks ◽  
Conversion Event ◽  
Yeast Mating ◽  
Mating Type Switching

Double-strand breaks in DNA are known to promote recombination in Saccharomyces cerevisiae. Yeast mating type switching, which is a highly efficient gene conversion event, is apparently initiated by a site-specific double-strand break. The 2 micrograms circle site-specific recombinase, FLP, has been shown to make double-strand breaks in its substrate DNA. By using a hybrid 2 micrograms circle::Tn5 plasmid, a portion of which resembles, in its DNA organization, the active (MAT) and the silent (HML) yeast mating type loci, it is shown that FLP mediates a conversion event analogous to mating type switching. Whereas the FLP site-specific recombination is not dependent on the RAD52 gene product, the FLP-induced conversion is abolished in a rad52 background. The FLP-promoted conversion in vivo can be faithfully reproduced by making a double-stranded gap in vitro in the vicinity of the FLP site and allowing the gap to be repaired in vivo.

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