scholarly journals Characterization of gross genome rearrangements in Deinococcus radiodurans recA mutants

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jelena Repar ◽  
Davor Zahradka ◽  
Ivan Sović ◽  
Ksenija Zahradka
Keyword(s):  
Genome Stability ◽  
Deinococcus Radiodurans ◽  
Genome Rearrangements ◽  
Genomic Region ◽  
Insertion Sequences ◽  
Dna Double Strand Breaks ◽  
Recombination Mechanism ◽  
Large Deletions ◽  
Single Strand Annealing ◽  
Strand Annealing

AbstractGenome stability in radioresistant bacterium Deinococcus radiodurans depends on RecA, the main bacterial recombinase. Without RecA, gross genome rearrangements occur during repair of DNA double-strand breaks. Long repeated (insertion) sequences have been identified as hot spots for ectopic recombination leading to genome rearrangements, and single-strand annealing (SSA) postulated to be the most likely mechanism involved in this process. Here, we have sequenced five isolates of D. radiodurans recA mutant carrying gross genome rearrangements to precisely characterize the rearrangements and to elucidate the underlying repair mechanism. The detected rearrangements consisted of large deletions in chromosome II in all the sequenced recA isolates. The mechanism behind these deletions clearly differs from the classical SSA; it utilized short (4–11 bp) repeats as opposed to insertion sequences or other long repeats. Moreover, it worked over larger linear DNA distances from those previously tested. Our data are most compatible with alternative end-joining, a recombination mechanism that operates in eukaryotes, but is also found in Escherichia coli. Additionally, despite the recA isolates being preselected for different rearrangement patterns, all identified deletions were found to overlap in a 35 kb genomic region. We weigh the evidence for mechanistic vs. adaptive reasons for this phenomenon.

scholarly journals Mechanism for accurate, protein-assisted DNA annealing by Deinococcus radiodurans DdrB

2016 ◽  
Vol 113 (16) ◽  
pp. 4308-4313 ◽  
Author(s):  
Seiji N. Sugiman-Marangos ◽  
Yoni M. Weiss ◽  
Murray S. Junop
Keyword(s):  
Deinococcus Radiodurans ◽  
Double Strand Breaks ◽  
Single Strand ◽  
High Fidelity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Damage Response ◽  
Single Strand Annealing ◽  
Dna Strands ◽  
Strand Annealing

Accurate pairing of DNA strands is essential for repair of DNA double-strand breaks (DSBs). How cells achieve accurate annealing when large regions of single-strand DNA are unpaired has remained unclear despite many efforts focused on understanding proteins, which mediate this process. Here we report the crystal structure of a single-strand annealing protein [DdrB (DNA damage response B)] in complex with a partially annealed DNA intermediate to 2.2 Å. This structure and supporting biochemical data reveal a mechanism for accurate annealing involving DdrB-mediated proofreading of strand complementarity. DdrB promotes high-fidelity annealing by constraining specific bases from unauthorized association and only releases annealed duplex when bound strands are fully complementary. To our knowledge, this mechanism provides the first understanding for how cells achieve accurate, protein-assisted strand annealing under biological conditions that would otherwise favor misannealing.

scholarly journals Necessities in the Processing of DNA Double Strand Breaks and Their Effects on Genomic Instability and Cancer

Cancers ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1671 ◽  
Author(s):  
George Iliakis ◽  
Emil Mladenov ◽  
Veronika Mladenova
Keyword(s):  
High Speed ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Large Deletions ◽  
Single Strand Annealing ◽  
Modes Of Processing ◽  
Non Homologous End Joining ◽  
Strand Annealing

Double strand breaks (DSBs) are induced in the DNA following exposure of cells to ionizing radiation (IR) and are highly consequential for genome integrity, requiring highly specialized modes of processing. Erroneous processing of DSBs is a cause of cell death or its transformation to a cancer cell. Four mechanistically distinct pathways have evolved in cells of higher eukaryotes to process DSBs, providing thus multiple options for the damaged cells. The homologous recombination repair (HRR) dependent subway of gene conversion (GC) removes IR-induced DSBs from the genome in an error-free manner. Classical non-homologous end joining (c-NHEJ) removes DSBs with very high speed but is unable to restore the sequence at the generated junction and can catalyze the formation of translocations. Alternative end-joining (alt-EJ) operates on similar principles as c-NHEJ but is slower and more error-prone regarding both sequence preservation and translocation formation. Finally, single strand annealing (SSA) is associated with large deletions and may also form translocations. Thus, the four pathways available for the processing of DSBs are not alternative options producing equivalent outcomes. We discuss the rationale for the evolution of pathways with such divergent properties and fidelities and outline the logic and necessities that govern their engagement. We reason that cells are not free to choose one specific pathway for the processing of a DSB but rather that they engage a pathway by applying the logic of highest fidelity selection, adapted to necessities imposed by the character of the DSB being processed. We introduce DSB clusters as a particularly consequential form of chromatin breakage and review findings suggesting that this form of damage underpins the increased efficacy of high linear energy transfer (LET) radiation modalities. The concepts developed have implications for the protection of humans from radon-induced cancer, as well as the treatment of cancer with radiations of high LET.

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 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.

scholarly journals RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair

2018 ◽  
Vol 115 (35) ◽  
pp. E8286-E8295 ◽  
Author(s):  
Liwei An ◽  
Chao Dong ◽  
Junshi Li ◽  
Jie Chen ◽  
Jingsong Yuan ◽  
...  
Keyword(s):  
Single Strand ◽  
Fine Tuning ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Single Strand Annealing ◽  
Molecular Rheostat ◽  
Dna End Resection ◽  
Strand Annealing ◽  
End Resection

Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSI-induced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, we found that RNF169 promotes CtIP-dependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates single-strand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways.

scholarly journals Single Strand Annealing Plays a Major Role in RecA-Independent Recombination between Repeated Sequences in the Radioresistant Deinococcus radiodurans Bacterium

PLoS Genetics ◽  
2015 ◽  
Vol 11 (10) ◽  
pp. e1005636 ◽  
Author(s):  
Solenne Ithurbide ◽  
Esma Bentchikou ◽  
Geneviève Coste ◽  
Bruno Bost ◽  
Pascale Servant ◽  
...  
Keyword(s):  
Deinococcus Radiodurans ◽  
Single Strand ◽  
Repeated Sequences ◽  
Single Strand Annealing ◽  
Strand Annealing

scholarly journals FKBP25 participates in DNA double-strand break repair

2020 ◽  
Vol 98 (1) ◽  
pp. 42-49 ◽  
Author(s):  
David Dilworth ◽  
Fade Gong ◽  
Kyle Miller ◽  
Christopher J. Nelson
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Peptide Bonds ◽  
Single Strand Annealing ◽  
Strand Annealing ◽  
Fk506 Binding Proteins

FK506-binding proteins (FKBPs) alter the conformation of proteins via cis–trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25’s catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.

scholarly journals Mutational signatures reveal the role of RAD52 in p53-independent p21 driven genomic instability

2017 ◽  
Author(s):  
Panagiotis Galanos ◽  
George Pappas ◽  
Alexander Polyzos ◽  
Athanassios Kotsinas ◽  
Ioanna Svolaki ◽  
...  
Keyword(s):  
Genomic Instability ◽  
Repair Process ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Nucleotide Substitutions ◽  
Mutational Signatures ◽  
Repair Capacity ◽  
Single Strand Annealing ◽  
Mechanistic Basis ◽  
Strand Annealing

AbstractBackgroundGenomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential to design appropriate therapeutic strategies. In a recent study we reported an unexpected oncogenic property of p21WAF1/Cip1 showing that its chronic expression, in a p53-deficient environment, causes genomic instability by deregulating the replication licensing machinery.ResultsExtending on this work we now demonstrate that p21WAF1/Cip1 can further fuel genomic instability by suppressing the repair capacity of low and high fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we found at the mechanistic level that the DSBs were repaired by Rad52-dependent Break-Induced Replication (BIR) and Single-Strand Annealing (SSA). Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route was deficient. Surprisingly, Rad52 was activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation.ConclusionsOur results signify the importance of mutational signatures as guides to disclose the “repair history” leading to genomic instability. In this vein, following this approach we unveiled how chronic p21WAF1/Cip1 expression rewires the repair process, identifying Rad52 as a source of genomic instability and a candidate therapeutic target.

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