The canonical non-homologous end joining factor XLF promotes chromosomal deletion rearrangements in human cells

Bhargava, Ragini; Lopezcolorado, Felicia Wednesday; Tsai, L. Jillianne; Stark, Jeremy M.

doi:10.1074/jbc.ra119.010421

Cited by 13 publications

(28 citation statements)

References 56 publications

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“…NHEJ competes with HR for access to DSBs [ 26 , 50 , 51 ], suggesting that NHEJ may occur more frequently in cells expressing β-HPV 8E6. To test this, we measured NHEJ efficiency, using a recently described end-joining assay that uses CD4 expression as a readout [ 28 ]. In this assay, CAS9 endonucleases are used to create breaks in the human genome downstream of the GAPDH promoter, and upstream of the CD4 exon.…”

Section: Resultsmentioning

confidence: 99%

“…As previously reported, expression of CAS9 endonucleases targeting GAPDH and CD4 lead to CD4 expression. The assay was further verified by treating U2OS cells with 10 μM ATM inhibitor (KU55933) and 10 μM DNA-PKcs inhibitor (NU7441), that are known to increase and decrease NHEJ, respectively [ 28 , 38 ]. As expected, KU55933 increased CD4 expression, while NU7441 decreased it ( Figure 1 B,C).…”

Section: Resultsmentioning

confidence: 99%

“…This occurs downstream of Artemis activation and is required for the DNA ligation step in NHEJ [ 49 , 61 , 62 ]. Like Artemis, XRCC4 is also a substrate of DNA-PKcs [ 27 , 28 ]. However, the role of that phosphorylation is poorly understood [ 63 , 64 ].…”

Section: Resultsmentioning

confidence: 99%

“…When HR fails, non-homologous end joining (NHEJ) serves as a backup repair mechanism. NHEJ is not restricted to any portion of the cell cycle, but tends to occur when HR is not available (i.e., G1 and early S phases) [ 26 , 27 , 28 , 29 , 30 ]. It is an error-prone pathway that initiates with 53BP1 binding to the DSB [ 31 , 32 ].…”

Section: Introductionmentioning

confidence: 99%

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Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity

Bugbee

Gamez

et al. 2020

Cancers

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Cutaneous viral infections occur in a background of near continual exposure to environmental genotoxins, like UV radiation in sunlight. Failure to repair damaged DNA is an established driver of tumorigenesis and substantial cellular resources are devoted to repairing DNA lesions. Beta-human papillomaviruses (β-HPVs) attenuate DNA repair signaling. However, their role in human disease is unclear. Some have proposed that β-HPV promotes tumorigenesis, while others suggest that β-HPV protects against skin cancer. Most of the molecular evidence that β-HPV impairs DNA repair has been gained via characterization of the E6 protein from β-HPV 8 (β-HPV 8E6). Moreover, β-HPV 8E6 hinders DNA repair by binding and destabilizing p300, a transcription factor for multiple DNA repair genes. By reducing p300 availability, β-HPV 8E6 attenuates a major double strand DNA break (DSB) repair pathway, homologous recombination. Here, β-HPV 8E6 impairs another DSB repair pathway, non-homologous end joining (NHEJ). Specifically, β-HPV 8E6 acts by attenuating DNA-dependent protein kinase (DNA-PK) activity, a critical NHEJ kinase. This includes DNA-PK activation and the downstream of steps in the pathway associated with DNA-PK activity. Notably, β-HPV 8E6 inhibits NHEJ through p300 dependent and independent means. Together, these data expand the known genome destabilizing capabilities of β-HPV 8E6.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity

Bugbee

Gamez

et al. 2020

Cancers

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“…These competing functions suggest that β-HPV8 E6 makes more mutagenic DSB repair in general and more specifically will increase the frequency of deletions. To test this, HFK LXSN and HFK β-HPV8 E6 cells were transfected with vectors that expressed CAS9 endonuclease and sgRNA to induces a DSB just upstream of the CD4 open reading frame [9,59]. A series of overlapping primers targeting the 100 Kb region upstream and downstream of the CAS9 target site were designed (Fig 8A), pooled and used to produce amplicons for next-generation sequencing.…”

Section: Beta-hpv 8 E6 Increases Genomic Instability During Dsb Repairmentioning

confidence: 99%

Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors

Wallace

Chen

Bugbee

et al. 2021

Preprint

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Beta human papillomavirus (β-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks in DNA (DSBs) are the most deleterious DNA lesion. They are typically repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). HR occurs after DNA replication while NHEJ can occur at any point in the cell cycle. They are not thought to occur in the same cell at the same time. By destabilizing p300, β-HPV type 8 protein E6 (β-HPV8 E6) attenuates both repair pathways. However, β-HPV8 E6 delays rather than abrogates DSB repair. Thus, β-HPV8 E6 may cause DSBs to be repaired through a more mutagenic process. To evaluate this, immunofluorescence microscopy was used to detect colocalization, formation, and resolution of DSB repair complexes following damage. Flow cytometry and immunofluorescence microscopy were used to determine the cell cycle distribution of repair complexes. The resulting data show that β-HPV8 E6 causes HR factors (RPA70 and RAD51) to colocalize with a persistent NHEJ factor (pDNA-PKcs). RPA70 complexes gave way to RAD51 complexes as in canonical HR, but RAD51 and pDNA-PKcs colocalization did not resolve within 32 hours of damage. The persistent RAD51 foci occur in G1 phase, consistent with recruitment after NHEJ fails. Chemical inhibition of p300, p300 knockout cells, and an β-HPV8 E6 mutant demonstrate that these phenotypes are the result of β-HPV8 E6-meidated p300 destabilization. Mutations associated with DSB repair were identified using next generation sequencing after a CAS9-induced DSB. β-HPV8 E6 increases the frequency of mutations (>15 fold) and deletions (>20 fold) associated with DSB repair. These data suggest that β-HPV8 E6 causes abnormal DSB repair where both NHEJ and HR occur at the same lesion and that his leads to deletions as the single stranded DNA produced during HR is removed by NHEJ.

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To indel or not to indel: Factors influencing mutagenesis during chromosomal break end joining

2022

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The canonical non-homologous end joining factor XLF promotes chromosomal deletion rearrangements in human cells

Cited by 13 publications

References 56 publications

Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity

Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity

Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors

To indel or not to indel: Factors influencing mutagenesis during chromosomal break end joining

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