Dynamic Processing of Displacement Loops During Recombinational DNA Repair

AbstractDisplacement-loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loopsin vivo, which enabled studying the kinetics of their formation and defining the network of D-loop formation and reversal pathways. Nascent D-loops are detected within 2 hrs of DSB formation and extended over the next 2 hrs in a system allowing break-induced replication. The majority of D-loops are disrupted in wild type cells by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a novel layer of HR control in cells relying on nascent D-loop dynamics, revealing unsuspected complexities, and identifying a surprising role for a conserved Rad54 paralog.

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Topological DNA-binding of structural maintenance of chromosomes-like RecN promotes DNA double-strand break repair in Escherichia coli

Communications Biology ◽

10.1038/s42003-019-0655-4 ◽

2019 ◽

Vol 2 (1) ◽

Cited By ~ 2

Author(s):

Kenji Keyamura ◽

Takashi Hishida

Keyword(s):

Escherichia Coli ◽

Strand Break ◽

Dependent Manner ◽

Loop Formation ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Dna Double Strand Break ◽

D Loop ◽

Linear Dsdna ◽

Structural Maintenance Of Chromosomes

AbstractBacterial RecN, closely related to the structural maintenance of chromosomes (SMC) family of proteins, functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination. Here we show that the purified Escherichia coli RecN protein topologically loads onto both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) that has a preference for ssDNA. RecN topologically bound to dsDNA slides off the end of linear dsDNA, but this is prevented by RecA nucleoprotein filaments on ssDNA, thereby allowing RecN to translocate to DSBs. Furthermore, we found that, once RecN is recruited onto ssDNA, it can topologically capture a second dsDNA substrate in an ATP-dependent manner, suggesting a role in synapsis. Indeed, RecN stimulates RecA-mediated D-loop formation and subsequent strand exchange activities. Our findings provide mechanistic insights into the recruitment of RecN to DSBs and sister chromatid interactions by RecN, both of which function in RecA-mediated DSB repair.

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The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo

Blood ◽

10.1182/blood-2010-01-265769 ◽

2010 ◽

Vol 116 (22) ◽

pp. 4578-4587 ◽

Cited By ~ 189

Author(s):

Victoria J. Weston ◽

Ceri E. Oldreive ◽

Anna Skowronska ◽

David G. Oscier ◽

Guy Pratt ◽

...

Keyword(s):

Cell Line ◽

Tumor Cells ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Prolymphocytic Leukemia ◽

Dna Double Strand Break ◽

Break Repair

Abstract The Ataxia Telangiectasia Mutated (ATM) gene is frequently inactivated in lymphoid malignancies such as chronic lymphocytic leukemia (CLL), T-prolymphocytic leukemia (T-PLL), and mantle cell lymphoma (MCL) and is associated with defective apoptosis in response to alkylating agents and purine analogues. ATM mutant cells exhibit impaired DNA double strand break repair. Poly (ADP-ribose) polymerase (PARP) inhibition that imposes the requirement for DNA double strand break repair should selectively sensitize ATM-deficient tumor cells to killing. We investigated in vitro sensitivity to the poly (ADP-ribose) polymerase inhibitor olaparib (AZD2281) of 5 ATM mutant lymphoblastoid cell lines (LCL), an ATM mutant MCL cell line, an ATM knockdown PGA CLL cell line, and 9 ATM-deficient primary CLLs induced to cycle and observed differential killing compared with ATM wildtype counterparts. Pharmacologic inhibition of ATM and ATM knockdown confirmed the effect was ATM-dependent and mediated through mitotic catastrophe independently of apoptosis. A nonobese diabetic/severe combined immunodeficient (NOD/SCID) murine xenograft model of an ATM mutant MCL cell line demonstrated significantly reduced tumor load and an increased survival of animals after olaparib treatment in vivo. Addition of olaparib sensitized ATM null tumor cells to DNA-damaging agents. We suggest that olaparib would be an appropriate agent for treating refractory ATM mutant lymphoid tumors.

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Repair Kinetics of Genomic Interstrand DNA Cross-Links: Evidence for DNA Double-Strand Break-Dependent Activation of the Fanconi Anemia/BRCA Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.24.1.123-134.2004 ◽

2004 ◽

Vol 24 (1) ◽

pp. 123-134 ◽

Cited By ~ 170

Author(s):

Andreas Rothfuss ◽

Markus Grompe

Keyword(s):

Fanconi Anemia ◽

Strand Break ◽

S Phase ◽

Dna Double Strand Breaks ◽

Subsequent Formation ◽

Strand Breaks ◽

Dna Double Strand Break ◽

Cross Links ◽

Kinetics Of

ABSTRACT The detailed mechanisms of DNA interstrand cross-link (ICL) repair and the involvement of the Fanconi anemia (FA)/BRCA pathway in this process are not known. Present models suggest that recognition and repair of ICL in human cells occur primarily during the S phase. Here we provide evidence for a refined model in which ICLs are recognized and are rapidly incised by ERCC1/XPF independent of DNA replication. However, the incised ICLs are then processed further and DNA double-strand breaks (DSB) form exclusively in the S phase. FA cells are fully proficient in the sensing and incision of ICL as well as in the subsequent formation of DSB, suggesting a role of the FA/BRCA pathway downstream in ICL repair. In fact, activation of FANCD2 occurs slowly after ICL treatment and correlates with the appearance of DSB in the S phase. In contrast, activation is rapid after ionizing radiation, indicating that the FA/BRCA pathway is specifically activated upon DSB formation. Furthermore, the formation of FANCD2 foci is restricted to a subpopulation of cells, which can be labeled by bromodeoxyuridine incorporation. We therefore conclude that the FA/BRCA pathway, while being dispensable for the early events in ICL repair, is activated in S-phase cells after DSB have formed.

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mus309 mutation, defective in DNA double-strand break repair, affects intergenic but not intragenic meiotic recombination in Drosophila melanogaster

Genetics Research ◽

10.1017/s0016672305007883 ◽

2005 ◽

Vol 86 (3) ◽

pp. 185-191 ◽

Cited By ~ 10

Author(s):

PETTER PORTIN

Keyword(s):

X Chromosome ◽

Meiotic Recombination ◽

Strand Break ◽

Double Strand Break ◽

Double Strand Break Repair ◽

Crossing Over ◽

Dna Double Strand Break ◽

D Loop ◽

Break Repair ◽

Strand Annealing

The effect was investigated of the hypomorphic DNA double-strand break repair, notably synthesis-dependent strand annealing, deficient mutation mus309 on the third chromosome of Drosophila melanogaster on intergenic and intragenic meiotic recombination in the X chromosome. The results showed that the mutation significantly increases the frequency of intergenic crossing over in two of three gene intervals of the X chromosome studied. Interestingly the increase was most prevalent in the tip of the X chromosome where crossovers normally are least frequent per physical map unit length. In particular crossing over interference was also affected, indicating that the effect of the mus309 mutation involves preconditions of crossing over but not the event of crossing over itself. On the other hand, the results also show that most probably the mutation does not have any effect on intragenic recombination, i.e. gene conversion. These results are fully consistent with the present molecular models of meiotic crossing over initiated by double-strand breaks of DNA followed by formation of a single-end-invasion intermediate, or D-loop, which is subsequently processed to generate either crossover or non-crossover products involving formation of a double Holliday junction. In particular the results suggest that the mus309 gene is involved in resolution of the D-loop, thereby affecting the choice between double-strand-break repair (DSBR) and synthesis-dependent strand annealing (SDSA) pathways of meiotic recombination.

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DNA double-strand break repair: a theoretical framework and its application

Journal of The Royal Society Interface ◽

10.1098/rsif.2015.0679 ◽

2016 ◽

Vol 13 (114) ◽

pp. 20150679 ◽

Cited By ~ 9

Author(s):

Philip J. Murray ◽

Bart Cornelissen ◽

Katherine A. Vallis ◽

S. Jon Chapman

Keyword(s):

Dna Damage ◽

Auger Electron ◽

Strand Break ◽

Dna Double Strand Breaks ◽

Dsb Repair ◽

Histone H2ax ◽

Dna Double Strand Break ◽

A Cell

DNA double-strand breaks (DSBs) are formed as a result of genotoxic insults, such as exogenous ionizing radiation, and are among the most serious types of DNA damage. One of the earliest molecular responses following DSB formation is the phosphorylation of the histone H2AX, giving rise to γ H2AX. Many copies of γ H2AX are generated at DSBs and can be detected in vitro as foci using well-established immuno-histochemical methods. It has previously been shown that anti- γ H2AX antibodies, modified by the addition of the cell-penetrating peptide TAT and a fluorescent or radionuclide label, can be used to visualize and quantify DSBs in vivo . Moreover, when labelled with a high amount of the short-range, Auger electron-emitting radioisotope, 111 In, the amount of DNA damage within a cell can be increased, leading to cell death. In this report, we develop a mathematical model that describes how molecular processes at individual sites of DNA damage give rise to quantifiable foci. Equations that describe stochastic mean behaviours at individual DSB sites are derived and parametrized using population-scale, time-series measurements from two different cancer cell lines. The model is used to examine two case studies in which the introduction of an antibody (anti- γ H2AX-TAT) that targets a key component in the DSB repair pathway influences system behaviour. We investigate: (i) how the interaction between anti- γ H2AX-TAT and γ H2AX effects the kinetics of H2AX phosphorylation and DSB repair and (ii) model behaviour when the anti- γ H2AX antibody is labelled with Auger electron-emitting 111 In and can thus instigate additional DNA damage. This work supports the conclusion that DSB kinetics are largely unaffected by the introduction of the anti- γ H2AX antibody, a result that has been validated experimentally, and hence the hypothesis that the use of anti- γ H2AX antibody to quantify DSBs does not violate the image tracer principle. Moreover, it provides a novel model of DNA damage accumulation in the presence of Auger electron-emitting 111 In that is supported qualitatively by the available experimental data.

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The balancing act of R-loop biology: The good, the bad, and the ugly

Journal of Biological Chemistry ◽

10.1074/jbc.rev119.011353 ◽

2019 ◽

Vol 295 (4) ◽

pp. 905-913 ◽

Cited By ~ 8

Author(s):

Youssef A. Hegazy ◽

Chrishan M. Fernando ◽

Elizabeth J. Tran

Keyword(s):

Genome Instability ◽

Strand Break ◽

Biological Processes ◽

Physiological Importance ◽

Future Goals ◽

Dna Double Strand Break ◽

Positive Functions ◽

Acid Structure

An R-loop is a three-stranded nucleic acid structure that consists of a DNA:RNA hybrid and a displaced strand of DNA. R-loops occur frequently in genomes and have significant physiological importance. They play vital roles in regulating gene expression, DNA replication, and DNA and histone modifications. Several studies have uncovered that R-loops contribute to fundamental biological processes in various organisms. Paradoxically, although they do play essential positive functions required for important biological processes, they can also contribute to DNA damage and genome instability. Recent evidence suggests that R-loops are involved in a number of human diseases, including neurological disorders, cancer, and autoimmune diseases. This review focuses on the molecular basis for R-loop–mediated gene regulation and genomic instability and briefly discusses methods for identifying R-loops in vivo. It also highlights recent studies indicating the role of R-loops in DNA double-strand break repair with an updated view of much-needed future goals in R-loop biology.

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Measurement of the Kinetics of DNA Double Strand Break Repair in Ehrlich Ascites Tumour Cells Using the Unwinding Method

International Journal of Radiation Biology and Related Studies in Physics Chemistry and Medicine ◽

10.1080/09553008014551691 ◽

1980 ◽

Vol 38 (3) ◽

pp. 335-347 ◽

Cited By ~ 38

Author(s):

P.E. Bryant ◽

D. Blöcher

Keyword(s):

Strand Break ◽

Tumour Cells ◽

Double Strand Break Repair ◽

Ascites Tumour ◽

Dna Double Strand Break ◽

Ehrlich Ascites ◽

Ehrlich Ascites Tumour ◽

Kinetics Of ◽

Ascites Tumour Cells ◽

Ehrlich Ascites Tumour Cells

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Active Role for Nibrin in the Kinetics of Atm Activation

Molecular and Cellular Biology ◽

10.1128/mcb.26.5.1691-1699.2006 ◽

2006 ◽

Vol 26 (5) ◽

pp. 1691-1699 ◽

Cited By ~ 62

Author(s):

Karen Cerosaletti ◽

Jocyndra Wright ◽

Patrick Concannon

Keyword(s):

Protein Kinase ◽

Binding Sites ◽

Mammalian Cells ◽

Active Role ◽

Nijmegen Breakage Syndrome ◽

Strand Break ◽

Wild Type ◽

Dna Double Strand Break ◽

Atm Protein ◽

Kinetics Of

ABSTRACT The Atm protein kinase is central to the DNA double-strand break response in mammalian cells. After irradiation, dimeric Atm undergoes autophosphorylation at Ser 1981 and dissociates into active monomers. Atm activation is stimulated by expression of the Mre11/Rad50/nibrin complex. Previously, we showed that a C-terminal fragment of nibrin, containing binding sites for both Mre11 and Atm, was sufficient to provide this stimulatory effect in Nijmegen breakage syndrome (NBS) cells. To discriminate whether nibrin's role in Atm activation is to bind and translocate Mre11/Rad50 to the nucleus or to interact directly with Atm, we expressed an Mre11 transgene with a C-terminal NLS sequence in NBS fibroblasts. The Mre11-NLS protein complexed with Rad50, localized to the nucleus in NBS fibroblasts, and associated with chromatin. However, Atm autophosphorylation was not stimulated in cells expressing Mre11-NLS, nor were downstream Atm targets phosphorylated. To determine whether nibrin-Atm interaction is necessary to stimulate Atm activation, we expressed nibrin transgenes lacking the Atm binding domain in NBS fibroblasts. The nibrin ΔAtm protein interacted with Mre11/Rad50; however, Atm autophosphorylation was dramatically reduced after irradiation in NBS cells expressing the nibrin ΔAtm transgenes relative to wild-type nibrin. These results indicate that nibrin plays an active role in Atm activation beyond translocating Mre11/Rad50 to the nucleus and that this function requires nibrin-Atm interaction.

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