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

Functional Role ◽

Excision Repair ◽

Double Strand Breaks ◽

Strand Breaks ◽

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.

Transcription-coupled DNA Double-Strand Breaks Are Mediated via the Nucleotide Excision Repair and the Mre11-Rad50-Nbs1 Complex

Molecular Biology of the Cell ◽

10.1091/mbc.e08-02-0215 ◽

2008 ◽

Vol 19 (9) ◽

pp. 3969-3981 ◽

Cited By ~ 51

Author(s):

Josée Guirouilh-Barbat ◽

Christophe Redon ◽

Yves Pommier

Keyword(s):

Excision Repair ◽

Double Strand Breaks ◽

Strand Breaks ◽

Cellular Effects ◽

Neutral Comet Assay ◽

Nucleotide Excision ◽

Full Activation ◽

Transcription And Replication

The cellular activity of Yondelis (trabectedin, Ecteinascidin 743, Et743) is known to depend on transcription-coupled nucleotide excision repair (TCR). However, the subsequent cellular effects of Et743 are not fully understood. Here we show that Et743 induces both transcription- and replication-coupled DNA double-strand breaks (DSBs) that are detectible by neutral COMET assay and as γ-H2AX foci that colocalize with 53BP1, Mre11, Ser1981-pATM, and Thr68-pChk2. The transcription coupled-DSBs (TC-DSBs) induced by Et743 depended both on TCR and Mre11-Rad50-Nbs1 (MRN) and were associated with DNA-PK–dependent γ-H2AX foci. In contrast to DNA-PK, ATM phosphorylated H2AX both in NER-proficient and -deficient cells, but its full activation was dependent on H2AX as well as DNA-PK, suggesting a positive feedback loop: DNA-PK-γ-H2AX-ATM. Knocking-out H2AX or inactivating DNA-PK reduced Et743's antiproliferative activity, whereas ATM and MRN tended to act as survival factors. Our results highlight the interplays between ATM and DNA-PK and their impacts on H2AX phosphorylation and cell survival. They also suggest that γ-H2AX may serve as a biomarker in patients treated with Et743 and that molecular profiling of tumors for TCR, MRN, ATM, and DNA-PK might be useful to anticipate tumor response to Et743 treatment.

P V.9 DNA double-strand breaks inhibit nucleotide excision repair by preventing formation of the protein repair complex at sites of damage

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis ◽

10.1016/s0027-5107(97)82715-7 ◽

1997 ◽

Vol 379 (1) ◽

pp. S36

Author(s):

Philippe Frit ◽

Patrick Calsou ◽

Bernard Salles

Keyword(s):

Excision Repair ◽

Double Strand Breaks ◽

Protein Repair ◽

Strand Breaks ◽

The role of DNA damage in laminopathy progeroid syndromes

Biochemical Society Transactions ◽

10.1042/bst20110700 ◽

2011 ◽

Vol 39 (6) ◽

pp. 1715-1718 ◽

Cited By ~ 19

Author(s):

Christopher J. Hutchison

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Excision Repair ◽

Double Strand Breaks ◽

Therapeutic Approaches ◽

Strand Breaks ◽

Progeroid Syndromes ◽

Repair Pathways

Progeroid laminopathies are characterized by the abnormal processing of lamin A, the appearance of misshapen nuclei, and the accumulation and persistence of DNA damage. In the present article, I consider the contribution of defective DNA damage pathways to the pathology of progeroid laminopathies. Defects in DNA repair pathways appear to be caused by a combination of factors. These include abnormal epigenetic modifications of chromatin that are required to recruit DNA repair pathways to sites of DNA damage, abnormal recruitment of DNA excision repair proteins to sites of DNA double-strand breaks, and unrepairable ROS (reactive oxygen species)-induced DNA damage. At least two of these defective processes offer the potential for novel therapeutic approaches.

H2AX phosphorylation within the G1 phase after UV irradiation depends on nucleotide excision repair and not DNA double-strand breaks

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0603779103 ◽

2006 ◽

Vol 103 (26) ◽

pp. 9891-9896 ◽

Cited By ~ 235

Author(s):

T. M. Marti ◽

E. Hefner ◽

L. Feeney ◽

V. Natale ◽

J. E. Cleaver

Keyword(s):

Uv Irradiation ◽

Excision Repair ◽

Double Strand Breaks ◽

G1 Phase ◽

H2ax Phosphorylation ◽

Strand Breaks ◽

The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage

Nucleic Acids Research ◽

10.1093/nar/27.16.3276 ◽

1999 ◽

Vol 27 (16) ◽

pp. 3276-3282 ◽

Cited By ~ 65

Author(s):

P. P. H. Van Sloun ◽

J. G. Jansen ◽

G. Weeda ◽

L. H. F. Mullenders ◽

A. A. van Zeeland ◽

...

Keyword(s):

Stem Cells ◽

Dna Damage ◽

Embryonic Stem Cells ◽

Excision Repair ◽

Embryonic Stem ◽

Genotoxic Effects ◽

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

Molecular and Cellular Biology ◽

10.1128/mcb.00156-17 ◽

2017 ◽

Vol 37 (24) ◽

Cited By ~ 8

Author(s):

Sucheta Arora ◽

Rajashree A. Deshpande ◽

Martin Budd ◽

Judy Campbell ◽

America Revere ◽

...

Keyword(s):

Nuclease Activity ◽

Double Strand Breaks ◽

Endonuclease Activity ◽

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.

Nucleotide Excision Repair Protein Rad23 Regulates Cell Virulence Independent of Rad4 in Candida albicans

mSphere ◽

10.1128/msphere.00062-20 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Jia Feng ◽

Shuangyan Yao ◽

Yansong Dong ◽

Jing Hu ◽

Malcolm Whiteway ◽

...

Keyword(s):

Dna Damage ◽

Candida Albicans ◽

Dna Damage Response ◽

Excision Repair ◽

Content Type ◽

Virulence Regulation ◽

Nucleotide Excision ◽

Damage Response

ABSTRACT In the pathogenic yeast Candida albicans, the DNA damage response contributes to pathogenicity by regulating cell morphology transitions and maintaining survival in response to DNA damage induced by reactive oxygen species (ROS) in host cells. However, the function of nucleotide excision repair (NER) in C. albicans has not been extensively investigated. To better understand the DNA damage response and its role in virulence, we studied the function of the Rad23 nucleotide excision repair protein in detail. The RAD23 deletion strain and overexpression strain both exhibit UV sensitivity, confirming the critical role of RAD23 in the nucleotide excision repair pathway. Genetic interaction assays revealed that the role of RAD23 in the UV response relies on RAD4 but is independent of RAD53, MMS22, and RAD18. RAD4 and RAD23 have similar roles in regulating cell morphogenesis and biofilm formation; however, only RAD23, but not RAD4, plays a negative role in virulence regulation in a mouse model. We found that the RAD23 deletion strain showed decreased survival in a Candida-macrophage interaction assay. Transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) data further revealed that RAD23, but not RAD4, regulates the transcription of a virulence factor, SUN41, suggesting a unique role of RAD23 in virulence regulation. Taking these observations together, our work reveals that the RAD23-related nucleotide excision pathway plays a critical role in the UV response but may not play a direct role in virulence. The virulence-related role of RAD23 may rely on the regulation of several virulence factors, which may give us further understanding about the linkage between DNA damage repair and virulence regulation in C. albicans. IMPORTANCE Candida albicans remains a significant threat to the lives of immunocompromised people. An understanding of the virulence and infection ability of C. albicans cells in the mammalian host may help with clinical treatment and drug discovery. The DNA damage response pathway is closely related to morphology regulation and virulence, as well as the ability to survive in host cells. In this study, we checked the role of the nucleotide excision repair (NER) pathway, the key repair system that functions to remove a large variety of DNA lesions such as those caused by UV light, but whose function has not been well studied in C. albicans. We found that Rad23, but not Rad4, plays a role in virulence that appears independent of the function of the NER pathway. Our research revealed that the NER pathway represented by Rad4/Rad23 may not play a direct role in virulence but that Rad23 may play a unique role in regulating the transcription of virulence genes that may contribute to the virulence of C. albicans.

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

Cell Death and Differentiation ◽

10.1038/s41418-019-0406-6 ◽

2019 ◽

Vol 27 (4) ◽

pp. 1200-1213 ◽

Cited By ~ 1

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 ◽

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.