characterizing-dna-repair-processes-at-transient-long-lasting-double

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

using-next-generation-sequencing-to-identify-mutations-associated

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication ...

Kansas State University

Given the high prevalence of cutaneous genus beta human papillomavirus (β-HPV) infections, it is important to understand how they manipulate their host cells. This is particularly true for cellular responses to UV damage, since our skin is continually exposed to UV. The E6 protein from β-genus HPV (β-HPV E6) decreases the abundance of two essential UV-repair kinases (ATM and ATR). Although β-HPV E6 reduces their availability, the impact on downstream signaling events is unclear. We demonstrate that β-HPV E6 decreases ATM and ATR activation. This inhibition extended to XPA, an ATR target necessary for UV repair, lowering both its phosphorylation and accumulation. β-HPV E6 also hindered POLη accumulation and foci formation, critical steps in translesion synthesis. ATM's phosphorylation of BRCA1 is also attenuated by β-HPV E6. While there was a striking decrease in phosphorylation of direct ATM/ATR targets, events further down the cascade were not reduced. In summary, despite being incomplete, β-HPV 8E6's hindrance of ATM/ATR has functional consequences.

β-HPV 8E6 Attenuates ATM and ATR Signaling in Response to UV Damage.

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.

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

Melanoma is a type of highly invasive skin cancer derived from melanocytes with poor prognosis. Vemurafenib (PLX4032) is a clinically approved targeted therapeutic for BRAF mutant melanoma that has a high therapeutic response rate and significantly prolongs the overall survival time of patients with melanoma. Antioxidants have been widely used as supplements for cancer prevention and for decreasing the side effects of cancer therapy. However, antioxidants can also protect cancer cells from oxidative stress and promote cancer growth and progression. The present study aimed to examine the effect of the antioxidants coenzyme Q10 (CoQ10) and β-carotene on melanoma cell growth and invasiveness and on the cytotoxicity of vemurafenib against both vemurafenib-sensitive (SK-MEL-28) and vemurafenib-resistant (A2058) human malignant melanoma cell lines. MTS assay and wound-healing assay demonstrated that CoQ10 alone significantly reduced the viability and migration of melanoma cells, respectively, and synergistically worked with vemurafenib to decrease the viability and migration of human melanoma cells. In contrast, MTS assay and flow cytometry revealed that β-carotene alone did not affect the viability and apoptosis induction of melanoma cells; however, it inhibited cell migration and invasiveness. Wound-healing and Transwell assay demonstrated that β-carotene alleviated the cytotoxicity of vemurafenib and mitigated the inhibitory effect of vemurafenib on cell migration and invasion. Both CoQ10 and β-carotene protected melanoma cells from undergoing apoptosis induced by vemurafenib. Immunoblotting demonstrated that β-carotene at physiological concentration worked synergistically with vemurafenib to suppress the Ras-Raf-Mek-Erk intracellular signaling pathway. The present study aimed to add to the evidence of the  effects of CoQ10 and β-carotene on the antimelanoma effects of vemurafenib.

Effect of antioxidants coenzyme Q10 and β-carotene on the cytotoxicity of vemurafenib against human malignant melanoma.

Beta human papillomavirus (β-HPV) are hypothesized to make DNA damage more mutagenic and potentially more carcinogenic. Double strand breaks (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. HR and NHEJ are not thought to occur in the same cell at the same time. HR is restricted to cells in phases of the cell cycle where homologous templates are available, while NHEJ occurs primarily during G1. β-HPV type 8 protein E6 (8E6) attenuates both repair pathways. We use a series of immunofluorescence microscopy and flow cytometry experiments to better define the impact of this attenuation. We found that 8E6 causes colocalization of HR factors (RPA70 and RAD51) with an NHEJ factor (activated DNA-PKcs or pDNA-PKcs) at persistent DSBs. 8E6 also causes RAD51 foci to form during G1. The initiation of NHEJ and HR at the same lesion could lead to antagonistic DNA end processing. Further, HR cannot be readily completed in an error-free manner during G1. Both aberrant repair events would cause deletions. To determine if these mutations were occurring, we used next generation sequencing of the 200kb surrounding a CAS9-induced DSB. 8E6 caused a 21-fold increase in deletions. Chemical and genetic inhibition of p300 as well as an 8E6 mutant that is incapable of destabilizing p300 demonstrates that 8E6 is acting via p300 destabilization. More specific chemical inhibitors of DNA repair provided mechanistic insight by mimicking 8E6-induced dysregulation of DNA repair in a virus-free system. Specifically, inhibition of NHEJ causes RAD51 foci to form in G1 and colocalization of RAD51 with pDNA-PKcs.

Changkun Hu

Division of Biology

Kansas State University

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter