scholarly journals Watching a double strand break repair polymerase insert a pro-mutagenic oxidized nucleotide

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
Akira Sassa ◽  
David D. Shock ◽  
William A. Beard ◽  
Samuel H. Wilson
Keyword(s):  
Active Site ◽  
Dna Polymerases ◽  
Time Lapse ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
X Ray Crystallography ◽  
Polymerase Fidelity ◽  
Break Repair

AbstractOxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (At). Rotation of At into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.

scholarly journals Second-Generation Antiandrogen Therapy Radiosensitizes Prostate Cancer Regardless of Castration State through Inhibition of DNA Double Strand Break Repair

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2467 ◽  
Author(s):  
Mohamed E. Elsesy ◽  
Su Jung Oh-Hohenhorst ◽  
Anastassia Löser ◽  
Christoph Oing ◽  
Sally Mutiara ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
First Generation ◽  
Second Generation ◽  
Strand Break ◽  
Double Strand Break ◽  
Abiraterone Acetate ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Castration Resistant ◽  
Break Repair

(1) Background: The combination of the first-generation antiandrogens and radiotherapy (RT) has been studied extensively in the clinical setting of prostate cancer (PCa). Here, we evaluated the potential radiosensitizing effect of the second-generation antiandrogens abiraterone acetate, apalutamide and enzalutamide. (2) Methods: Cell proliferation and agarose-colony forming assay were used to measure the effect on survival. Double strand break repair efficiency was monitored using immunofluorescence staining of γH2AX/53BP1. (3) Results: We report retrospectively a minor benefit for PCa patients received first-generation androgen blockers and RT compared to patients treated with RT alone. Combining either of the second-generation antiandrogens and 2Gy suppressed cell growth and increased doubling time significantly more than 2Gy alone, in both hormone-responsive LNCaP and castration-resistant C4-2B cells. These findings were recapitulated in resistant sub-clones to (i) hormone ablation (LNCaP-abl), (ii) abiraterone acetate (LNCaP-abi), (iii) apalutamide (LNCaP-ARN509), (iv) enzalutamide (C4-2B-ENZA), and in castration-resistant 22-RV1 cells. This radiosensitization effect was not observable using the first-generation antiandrogen bicalutamide. Inhibition of DNA DSB repair was found to contribute to the radiosensitization effect of second-generation antiandrogens, as demonstrated by a significant increase in residual γH2AX and 53BP1 foci numbers at 24h post-IR. DSB repair inhibition was further demonstrated in 22 patient-derived tumor slice cultures treated with abiraterone acetate before ex-vivo irradiation with 2Gy. (4) Conclusion: Together, these data show that second-generation antiandrogens can enhance radiosensitivity in PCa through DSB repair inhibition, regardless of their hormonal status. Translated into clinical practice, our results may help to find additional strategies to improve the effectiveness of RT in localized PCa, paving the way for a clinical trial.

scholarly journals Involvement of POLA2 in Double Strand Break Repair and Genotoxic Stress

2020 ◽  
Vol 21 (12) ◽  
pp. 4245
Author(s):  
Tuyen T. Dang ◽  
Julio C. Morales
Keyword(s):  
Dna Damage ◽  
Genomic Instability ◽  
Dna Polymerase ◽  
Genotoxic Stress ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Dna Polymerase Alpha ◽  
Break Repair

Cellular survival is dependent on the efficient replication and transmission of genomic information. DNA damage can be introduced into the genome by several different methods, one being the act of DNA replication. Replication is a potent source of DNA damage and genomic instability, especially through the formation of DNA double strand breaks (DSBs). DNA polymerase alpha is responsible for replication initiation. One subunit of the DNA polymerase alpha replication machinery is POLA2. Given the connection between replication and genomic instability, we decided to examine the role of POLA2 in DSB repair, as little is known about this topic. We found that loss of POLA2 leads to an increase in spontaneous DSB formation. Loss of POLA2 also slows DSB repair kinetics after treatment with etoposide and inhibits both of the major double strand break repair pathways: non-homologous end-joining and homologous recombination. In addition, loss of POLA2 leads to increased sensitivity to ionizing radiation and PARP1 inhibition. Lastly, POLA2 expression is elevated in glioblastoma multiforme tumors and correlates with poor overall patient survival. These data demonstrate a role for POLA2 in DSB repair and resistance to genotoxic stress.

Human INO80 chromatin-remodelling complex contributes to DNA double-strand break repair via the expression of Rad54B and XRCC3 genes

2010 ◽  
Vol 431 (2) ◽  
pp. 179-187 ◽  
Author(s):  
Eun-Jung Park ◽  
Shin-Kyoung Hur ◽  
Jongbum Kwon
Keyword(s):  
Chinese Hamster ◽  
Strand Break ◽  
Double Strand Break ◽  
Chromatin Remodelling ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Direct Role ◽  
Dna Double Strand Break ◽  
Break Repair ◽  
Repair Defect

Recent studies have shown that the SWI/SNF family of ATP-dependent chromatin-remodelling complexes play important roles in DNA repair as well as in transcription. The INO80 complex, the most recently described member of this family, has been shown in yeast to play direct role in DNA DSB (double-strand break) repair without affecting the expression of the genes involved in this process. However, whether this function of the INO80 complex is conserved in higher eukaryotes has not been investigated. In the present study, we found that knockdown of hINO80 (human INO80) confers DNA-damage hypersensitivity and inefficient DSB repair. Microarray analysis and other experiments have identified the Rad54B and XRCC3 (X-ray repair complementing defective repair in Chinese-hamster cells 3) genes, implicated in DSB repair, to be repressed by hINO80 deficiency. Chromatin immunoprecipitation studies have shown that hINO80 binds to the promoters of the Rad54B and XRCC3 genes. Re-expression of the Rad54B and XRCC3 genes rescues the DSB repair defect in hINO80-deficient cells. These results suggest that hINO80 assists DSB repair by positively regulating the expression of the Rad54B and XRCC3 genes. Therefore, unlike yeast INO80, hINO80 can contribute to DSB repair indirectly via gene expression, suggesting that the mechanistic role of this chromatin remodeller in DSB repair is evolutionarily diversified.

scholarly journals MOF and Histone H4 Acetylation at Lysine 16 Are Critical for DNA Damage Response and Double-Strand Break Repair

2010 ◽  
Vol 30 (14) ◽  
pp. 3582-3595 ◽  
Author(s):  
Girdhar G. Sharma ◽  
Sairei So ◽  
Arun Gupta ◽  
Rakesh Kumar ◽  
Christelle Cayrou ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Histone H4 ◽  
Dsb Repair ◽  
Altered Expression ◽  
Damage Response ◽  
Break Repair

ABSTRACT The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that reduced levels of H4K16ac correlate with a defective DNA damage response (DDR) and double-strand break (DSB) repair to ionizing radiation (IR). The defect, however, is not due to altered expression of proteins involved in DDR. Abrogation of IR-induced DDR by MOF depletion is inhibited by blocking H4K16ac deacetylation. MOF was found to be associated with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a protein involved in nonhomologous end-joining (NHEJ) repair. ATM-dependent IR-induced phosphorylation of DNA-PKcs was also abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased DNA double-strand break repair by both NHEJ and homologous recombination (HR). In addition, MOF activity was associated with general chromatin upon DNA damage and colocalized with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac (histone code), has a critical role at multiple stages in the cellular DNA damage response and DSB repair.

scholarly journals Expansions and Contractions in a Tandem Repeat Induced by Double-Strand Break Repair

1998 ◽  
Vol 18 (4) ◽  
pp. 2045-2054 ◽  
Author(s):  
Frédéric Pâques ◽  
Wai-Ying Leung ◽  
James E. Haber
Keyword(s):  
Tandem Repeat ◽  
Strand Break ◽  
Double Strand Break ◽  
Repeated Sequences ◽  
Tandem Array ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Break Repair ◽  
Strand Annealing ◽  
Donor Template

ABSTRACT Repair of a double-strand break (DSB) in yeast can induce very frequent expansions and contractions in a tandem array of 375-bp repeats. These results strongly suggest that DSB repair can be a major source of amplification of tandemly repeated sequences. Most of the DSB repair events are not associated with crossover. Rearrangements appear in 50% of these repaired recipient molecules. In contrast, the donor template nearly always remains unchanged. Among the rare crossover events, similar rearrangements are found. These results cannot readily be explained by the gap repair model of Szostak et al. (J. W. Szostak, T. L. Orr-Weaver, R. J. Rothstein, and F. W. Stahl, Cell 33:25–35, 1983) but can be explained by synthesis-dependent strand annealing (SDSA) models that allow for crossover. Support for SDSA models is provided by a demonstration that a single DSB repair event can use two donor templates located on two different chromosomes.

scholarly journals Time-lapse crystallography snapshots of a double-strand break repair polymerase in action

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Joonas A. Jamsen ◽  
William A. Beard ◽  
Lars C. Pedersen ◽  
David D. Shock ◽  
Andrea F. Moon ◽  
...  
Keyword(s):  
Time Lapse ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Break Repair

scholarly journals The human HELLS chromatin remodelling protein promotes end resection to facilitate homologous recombination and contributes to DSB repair within heterochromatin

2019 ◽  
Vol 48 (4) ◽  
pp. 1872-1885 ◽  
Author(s):  
Gabriel Kollárovič ◽  
Caitríona E Topping ◽  
Edward P Shaw ◽  
Anna L Chambers
Keyword(s):  
Homologous Recombination ◽  
Cancer Biology ◽  
Genome Stability ◽  
Strand Break ◽  
Double Strand Break ◽  
Chromatin Remodelling ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Break Repair ◽  
End Resection

Abstract Efficient double-strand break repair in eukaryotes requires manipulation of chromatin structure. ATP-dependent chromatin remodelling enzymes facilitate different DNA repair pathways, during different stages of the cell cycle and in varied chromatin environments. The contribution of remodelling factors to double-strand break repair within heterochromatin during G2 is unclear. The human HELLS protein is a Snf2-like chromatin remodeller family member and is mutated or misregulated in several cancers and some cases of ICF syndrome. HELLS has been implicated in the DNA damage response, but its mechanistic function in repair is not well understood. We discover that HELLS facilitates homologous recombination at two-ended breaks and contributes to repair within heterochromatic regions during G2. HELLS promotes initiation of HR by facilitating end-resection and accumulation of CtIP at IR-induced foci. We identify an interaction between HELLS and CtIP and establish that the ATPase domain of HELLS is required to promote DSB repair. This function of HELLS in maintenance of genome stability is likely to contribute to its role in cancer biology and demonstrates that different chromatin remodelling activities are required for efficient repair in specific genomic contexts.

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