scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

scholarly journals Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks

2007 ◽  
Vol 177 (2) ◽  
pp. 219-229 ◽  
Author(s):  
Naoya Uematsu ◽  
Eric Weterings ◽  
Ken-ichi Yano ◽  
Keiko Morotomi-Yano ◽  
Burkhard Jakob ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Laser System ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Dna Ends

The DNA-dependent protein kinase catalytic subunit (DNA-PKCS) plays an important role during the repair of DNA double-strand breaks (DSBs). It is recruited to DNA ends in the early stages of the nonhomologous end-joining (NHEJ) process, which mediates DSB repair. To study DNA-PKCS recruitment in vivo, we used a laser system to introduce DSBs in a specified region of the cell nucleus. We show that DNA-PKCS accumulates at DSB sites in a Ku80-dependent manner, and that neither the kinase activity nor the phosphorylation status of DNA-PKCS influences its initial accumulation. However, impairment of both of these functions results in deficient DSB repair and the maintained presence of DNA-PKCS at unrepaired DSBs. The use of photobleaching techniques allowed us to determine that the kinase activity and phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. We suggest a model in which DNA-PKCS phosphorylation/autophosphorylation facilitates NHEJ by destabilizing the interaction of DNA-PKCS with the DNA ends.

scholarly journals A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
William H. Gittens ◽  
Dominic J. Johnson ◽  
Rachal M. Allison ◽  
Tim J. Cooper ◽  
Holly Thomas ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Genome Stability ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Human Genomes ◽  
Nucleotide Resolution

Abstract DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

scholarly journals Role of the Nuclease Activity ofSaccharomyces cerevisiaeMre11 in Repair of DNA Double-Strand Breaks in Mitotic Cells

Genetics ◽  
2004 ◽  
Vol 166 (4) ◽  
pp. 1701-1713 ◽  
Author(s):  
L Kevin Lewis ◽  
Francesca Storici ◽  
Stephen Van Komen ◽  
Shanna Calero ◽  
Patrick Sung ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Complex Functions ◽  
Mitotic Cells

AbstractThe Rad50:Mre11:Xrs2 (RMX) complex functions in repair of DNA double-strand breaks (DSBs) by recombination and nonhomologous end-joining (NHEJ) and is also required for telomere stability. The Mre11 subunit exhibits nuclease activities in vitro, but the role of these activities in repair in mitotic cells has not been established. In this study we have performed a comparative study of three mutants (mre11-D16A, -D56N, and -H125N) previously shown to have reduced nuclease activities in vitro. In ends-in and ends-out chromosome recombination assays using defined plasmid and oligonucleotide DNA substrates, mre11-D16A cells were as deficient as mre11 null strains, but defects were small in mre11-D56N and -H125N mutants. mre11-D16A cells, but not the other mutants, also displayed strong sensitivity to ionizing radiation, with residual resistance largely dependent on the presence of the partially redundant nuclease Exo1. mre11-D16A mutants were also most sensitive to the S-phase-dependent clastogens hydroxyurea and methyl methanesulfonate but, as previously observed for D56N and H125N mutants, were not defective in NHEJ. Importantly, the affinity of purified Mre11-D16A protein for Rad50 and Xrs2 was indistinguishable from wild type and the mutant protein formed complexes with equivalent stoichiometry. Although the role of the nuclease activity has been questioned in previous studies, the comparative data presented here suggest that the nuclease function of Mre11 is required for RMX-mediated recombinational repair and telomere stabilization in mitotic cells.

scholarly journals Stepwise 5′ DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble

2019 ◽  
Vol 116 (12) ◽  
pp. 5505-5513 ◽  
Author(s):  
Elda Cannavo ◽  
Giordano Reginato ◽  
Petr Cejka
Keyword(s):  
Atp Hydrolysis ◽  
Double Strand Breaks ◽  
Dependent Manner ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Dna Strands ◽  
Dna Strand ◽  
The Individual

To repair DNA double-strand breaks by homologous recombination, the 5′-terminated DNA strands must first be resected to produce 3′ overhangs. Mre11 fromSaccharomyces cerevisiaeis a 3′ → 5′ exonuclease that is responsible for 5′ end degradation in vivo. Using plasmid-length DNA substrates and purified recombinant proteins, we show that the combined exonuclease and endonuclease activities of recombinant MRX-Sae2 preferentially degrade the 5′-terminated DNA strand, which extends beyond the vicinity of the DNA end. Mechanistically, Rad50 restricts the Mre11 exonuclease in an ATP binding-dependent manner, preventing 3′ end degradation. Phosphorylated Sae2, along with stimulating the MRX endonuclease as shown previously, also overcomes this inhibition to promote the 3′ → 5′ exonuclease of MRX, which requires ATP hydrolysis by Rad50. Our results support a model in which MRX-Sae2 catalyzes 5′-DNA end degradation by stepwise endonucleolytic DNA incisions, followed by exonucleolytic 3′ → 5′ degradation of the individual DNA fragments. This model explains how both exonuclease and endonuclease activities of Mre11 functionally integrate within the MRX-Sae2 ensemble to resect 5′-terminated DNA.

scholarly journals ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks

2020 ◽  
Vol 48 (17) ◽  
pp. 9710-9723
Author(s):  
Sébastien Britton ◽  
Pauline Chanut ◽  
Christine Delteil ◽  
Nadia Barboule ◽  
Philippe Frit ◽  
...  
Keyword(s):  
Dna Repair ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Repair Pathways ◽  
Non Homologous End Joining ◽  
Dna Strand ◽  
Dna Ends

Abstract Two DNA repair pathways operate at DNA double strand breaks (DSBs): non-homologous end-joining (NHEJ), that requires two adjacent DNA ends for ligation, and homologous recombination (HR), that resects one DNA strand for invasion of a homologous duplex. Faithful repair of replicative single-ended DSBs (seDSBs) is mediated by HR, due to the lack of a second DNA end for end-joining. ATM stimulates resection at such breaks through multiple mechanisms including CtIP phosphorylation, which also promotes removal of the DNA-ends sensor and NHEJ protein Ku. Here, using a new method for imaging the recruitment of the Ku partner DNA-PKcs at DSBs, we uncover an unanticipated role of ATM in removing DNA-PKcs from seDSBs in human cells. Phosphorylation of DNA-PKcs on the ABCDE cluster is necessary not only for DNA-PKcs clearance but also for the subsequent MRE11/CtIP-dependent release of Ku from these breaks. We propose that at seDSBs, ATM activity is necessary for the release of both Ku and DNA-PKcs components of the NHEJ apparatus, and thereby prevents subsequent aberrant interactions between seDSBs accompanied by DNA-PKcs autophosphorylation and detrimental commitment to Lig4-dependent end-joining.

scholarly journals A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

2019 ◽  
Author(s):  
William Gittens ◽  
Dominic J. Johnson ◽  
Rachal M. Allison ◽  
Tim J. Cooper ◽  
Holly Thomas ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Genome Stability ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Human Genomes ◽  
Nucleotide Resolution

AbstractDNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast, and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy enables us to reveal the influence primary DNA sequence has upon Top2 cleavage—distinguishing canonical DNA double-strand breaks (DSBs) from a major population of DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

scholarly journals The functional role of nucleotide excision repair in transcription-associated DNA damage in mammals

2019 ◽  
Author(s):  
Κυριάκος Αγαθαγγέλου
Keyword(s):  
Dna Damage ◽  
Nucleotide Excision Repair ◽  
Functional Role ◽  
Excision Repair ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Nucleotide Excision

Σε αντίθεση με τις πρωτεΐνες και τα υπόλοιπα μακρομόρια π.χ. τα σάκχαρα ή τα λίπη, το πυρηνικό DNA (η απαρχή του RNA και των πρωτεϊνών) είναι αναντικατάστατο. Παρά το γεγονός ότι η χημική του σύσταση είναι εξαιρετικά ασταθής, το DNA οφείλει να διατηρηθεί αναλλοίωτο καθόλη τη διάρκεια της ζωής του κυττάρου ώστε η γενετική πληροφορία να κληρονομηθεί αυτούσια στα θυγατρικά κύτταρα. Ωστόσο, η ύπαρξη διαφόρων ενδογενών γενοτοξικών παραγόντων προκαλούν τη σταδιακή συσσώρευση πλήθους δομικών αλλοιώσεων και προσβολών (π.χ. υδρόλυση, απαμίνωση βάσεων, διμερισμός πυριμιδινών, δημιουργία θραυσμάτων μονής ή διπλής DNA έλικας κτλ.) στο DNA. Η επακόλουθη γενωμική αστάθεια επιφέρει δραματικές αλλαγές στη φυσιολογία του κυττάρου παρεμποδίζοντας τη φυσιολογική λειτουργία ζωτικών βιολογικών διεργασιών όπως η μεταγραφή ή/και ο αναδιπλασιασμός του DNA. Για να αντιμετωπίσουν την σταδιακή συσσώρευση DNA βλαβών, τα ευκαρυωτικά κύτταρα έχουν αναπτύξει ένα σύνολο αλληλεπικαλυπτόμενων επιδιορθωτικών μηχανισμών, συμπεριλαμβανομένου του μηχανισμού εκτομής νουκλεοτιδίων (Nucleotide Excision Repair, NER), που εντοπίζουν, επιδιορθώνουν και αποκαθιστούν το προσβαλλόμενο DNA στην αρχική του μορφή. Στον άνθρωπο και τα αντίστοιχα πειραματικά μοντέλα ποντικών, η ύπαρξη εγγενών μεταλλαγών σε γονίδια του μονοπατιού NER, προκαλεί ένα ευρύ φάσμα κλινικών συμπτωμάτων που χαρακτηρίζεται από εξαιρετική ετερογένεια, η οποία δε μπορεί να εξηγηθεί αποκλειστικά λόγω της ατελούς επιδιόρθωσης του DNA. Πρόσφατες μελέτες απεκάλυψαν ότι ορισμένες πρωτεΐνες του NER συμμετέχουν, πέραν της επιδιόρθωσης των DNA βλαβών, σε κυτταρικές διεργασίες όπως η έναρξη της μεταγραφής και η αναδιαμόρφωση ή ο σχηματισμός της τρισδιάστατης δομής της χρωματίνης στο χώρο. Για να διαλευκάνουμε το λειτουργικό ρόλο του NER στην ανάπτυξη και τις ασθένειες στα θηλαστικά, αναπτύξαμε την μέθοδο της in vivo σήμανσης με βιοτίνη της πρωτεΐνης XPF στον ποντικό. Η προσέγγιση αυτή σε συνδυασμό με μεθοδολογίες αλληλούχισης DNA υψηλής απόδοσης και λειτουργικές προσεγγίσεις απεκάλυψαν ότι το ετεροδιμερές του συμπλόκου ενδονουκλεάσης του NER ERCCI-XPF αλληλεπιδρά με πρωτεϊνικούς παράγοντες που συμμετέχουν στην μεταγραφή και την εξομάλυνση του τοπολογικού φόρτου του DNA κατά την διαδικασία της μεταγραφής. Συγκεκριμένα, ανιχνεύσαμε ότι κατά την επαγωγή της μεταγραφής, η ERCC1-XPF προσδένεται σε υποκινητές, κατά μήκος του γονιδιώματος. Επιπλέον μελέτες απεκάλυψαν ότι η πρόσδεση του συμπλόκου ERCC1-XPF στο DNA, συμπίπτει με την δημιουργία εκτομών διπλού θραύσματος στο DNA (DNA double strand breaks, DSBs) σε διακριτές περιοχές του DNA. Τα αποτελέσματα της μελέτης αναδεικνύουν τον λειτουργικό ρόλο της ERCC1-XPF στην επιδιόρθωση DNA βλαβών που προκαλούνται κατά την διαδικασία της μεταγραφής και παρέχουν ένα μηχανιστικό μοντέλο που εξηγεί ικανοποιητικά την κλινική ετερογένεια των συνδρόμων NER.

scholarly journals Physiological Roles of DNA Double-Strand Breaks

2017 ◽  
Vol 2017 ◽  
pp. 1-20 ◽  
Author(s):  
Farhaan A. Khan ◽  
Syed O. Ali
Keyword(s):  
Double Helix ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Genomic Integrity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Strand Breaks ◽  
Spatiotemporal Regulation ◽  
Dna Double Strand Break

Genomic integrity is constantly threatened by sources of DNA damage, internal and external alike. Among the most cytotoxic lesions is the DNA double-strand break (DSB) which arises from the cleavage of both strands of the double helix. Cells boast a considerable set of defences to both prevent and repair these breaks and drugs which derail these processes represent an important category of anticancer therapeutics. And yet, bizarrely, cells deploy this very machinery for the intentional and calculated disruption of genomic integrity, harnessing potentially destructive DSBs in delicate genetic transactions. Under tight spatiotemporal regulation, DSBs serve as a tool for genetic modification, widely used across cellular biology to generate diverse functionalities, ranging from the fundamental upkeep of DNA replication, transcription, and the chromatin landscape to the diversification of immunity and the germline. Growing evidence points to a role of aberrant DSB physiology in human disease and an understanding of these processes may both inform the design of new therapeutic strategies and reduce off-target effects of existing drugs. Here, we review the wide-ranging roles of physiological DSBs and the emerging network of their multilateral regulation to consider how the cell is able to harness DNA breaks as a critical biochemical tool.

scholarly journals Helicobacter pylori Infection Introduces DNA Double-Strand Breaks in Host Cells

2014 ◽  
Vol 82 (10) ◽  
pp. 4182-4189 ◽  
Author(s):  
Katsuhiro Hanada ◽  
Tomohisa Uchida ◽  
Yoshiyuki Tsukamoto ◽  
Masahide Watada ◽  
Nahomi Yamaguchi ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
Genomic Instability ◽  
Double Strand Breaks ◽  
Host Cells ◽  
Dna Double Strand Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
H Pylori

ABSTRACTGastric cancer is an inflammation-related malignancy related to long-standing acute and chronic inflammation caused by infection with the human bacterial pathogenHelicobacter pylori. Inflammation can result in genomic instability. However, there are considerable data thatH. pyloriitself can also produce genomic instability both directly and through epigenetic pathways. Overall, the mechanisms ofH. pylori-induced host genomic instabilities remain poorly understood. We used microarray screening ofH. pylori-infected human gastric biopsy specimens to identify candidate genes involved inH. pylori-induced host genomic instabilities. We found upregulation ofATMexpressionin vivoin gastric mucosal cells infected withH. pylori. Using gastric cancer cell lines, we confirmed that theH. pylori-related activation of ATM was due to the accumulation of DNA double-strand breaks (DSBs). DSBs were observed following infection with bothcagpathogenicity island (PAI)-positive and -negative strains, but the effect was more robust withcagPAI-positive strains. These results are consistent with the fact that infections with bothcagPAI-positive and -negative strains are associated with gastric carcinogenesis, but the risk is higher in individuals infected withcagPAI-positive strains.

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