XRCC4 and MRE11 Roles and Transcriptional Response to Repair of TALEN-Induced Double-Strand DNA Breaks

Double-strand breaks (DSB) are one of the most lethal forms of DNA damage that, if left unrepaired, can lead to genomic instability, cellular transformation, and cell death. In this work, we examined how repair of transcription activator-like effector nuclease (TALEN)-induced DNA damage was altered when knocking out, or inhibiting a function of, two DNA repair proteins, XRCC4 and MRE11, respectively. We developed a fluorescent reporter assay that uses TALENs to introduce DSB and detected repair by the presence of GFP fluorescence. We observed repair of TALEN-induced breaks in the XRCC4 knockout cells treated with mirin (a pharmacological inhibitor of MRE11 exonuclease activity), albeit with ~40% reduced efficiency compared to normal cells. Editing in the absence of XRCC4 or MRE11 exonuclease was robust, with little difference between the indel profiles amongst any of the groups. Reviewing the transcriptional profiles of the mirin-treated XRCC4 knockout cells showed 307 uniquely differentially expressed genes, a number far greater than for either of the other cell lines (the HeLa XRCC4 knockout sample had 83 genes, and the mirin-treated HeLa cells had 30 genes uniquely differentially expressed). Pathways unique to the XRCC4 knockout+mirin group included differential expression of p53 downstream pathways, and metabolic pathways indicating cell adaptation for energy regulation and stress response. In conclusion, our study showed that TALEN-induced DSBs are repaired, even when a key DSB repair protein or protein function is not operational, without a change in indel profiles. However, transcriptional profiles indicate the induction of unique cellular responses dependent upon the DNA repair protein(s) hampered.

MV1035 Overcomes Temozolomide Resistance in Patient-Derived Glioblastoma Stem Cell Lines

Glioblastoma (GBM, grade IV glioma) represents the most aggressive brain tumor and patients with GBM have a poor prognosis. Until now surgical resection followed by radiotherapy and temozolomide (TMZ) treatment represents the standard strategy for GBM. We showed that the imidazobenzoxazin-5-thione MV1035 is able to significantly reduce GBM U87-MG cells migration and invasiveness through inhibition of the RNA demethylase ALKBH5. In this work, we focus on the DNA repair protein ALKBH2, a further MV1035 target resulting from SPILLO-PBSS proteome-wide scale in silico analysis. Our data demonstrate that MV1035 inhibits the activity of ALKBH2, known to be involved in GBM TMZ resistance. MV1035 was used on both U87-MG and two patient-derived (PD) glioma stem cells (GSCs): in combination with TMZ, it has a significant synergistic effect in reducing cell viability and sphere formation. Moreover, MV1035 induces a reduction in MGMT expression in PD-GSCs cell lines most likely through a mechanism that acts on MGMT promoter methylation. Taken together our data show that MV1035 could act as an inhibitor potentially helpful to overcome TMZ resistance and able to reduce GBM migration and invasiveness.

Is DNA repair controlled by a biological logic circuit?

The possible utilization of biological logic circuit(s) in the integration and regulation of DNA repair is discussed. The author believes this mode of regulation likely applies to many other areas of cell biology; however, there are currently more experimental data to support its involvement in the control of DNA repair. Sequential logic processes always require a clock to orchestrate the orderly processing of events. In the proposed hypothesis, the pulses in the expression of p53 serve this function. Given the many advantages of logic type control, one would expect that in the course of ~ 3 billion years of evolution, where single cell life forms were likely the only forms of life, a biological logic type control system would have evolved to control at least some biological processes. Several other required components in addition to the ‘clock’ have been identified, such as; a method to temporarily inactivate repair processes when they are not required (e.g. the reversible inactivation of MGMT, a suicide repair protein, by phosphorylation); this prevents complex DNA repair systems with potentially overlapping repair functions from interfering with each other.

Initiation and Prevention of Biological Damage by Radiation-Generated Protein Radicals

Ionizing radiations cause chemical damage to proteins. In aerobic aqueous solutions, the damage is commonly mediated by the hydroxyl free radicals generated from water, resulting in formation of protein radicals. Protein damage is especially significant in biological systems, because proteins are the most abundant targets of the radiation-generated radicals, the hydroxyl radical-protein reaction is fast, and the damage usually results in loss of their biological function. Under physiological conditions, proteins are initially oxidized to carbon-centered radicals, which can propagate the damage to other molecules. The most effective endogenous antioxidants, ascorbate, GSH, and urate, are unable to prevent all of the damage under the common condition of oxidative stress. In a promising development, recent work demonstrates the potential of polyphenols, their metabolites, and other aromatic compounds to repair protein radicals by the fast formation of less damaging radical adducts, thus potentially preventing the formation of a cascade of new reactive species.

ATM and RAD51 Repair Pathways in Human Lymphocytes Irradiated with 70 MeV Therapeutic Proton Beam

The repair of radiation-induced DNA damage is a key factor differentiating patients in terms of the therapeutic efficacy and toxicity to surrounding normal tissue. Proton energy substantially determines the types of cancers that can be treated. The present work investigated the DNA double-strand break repair systems, represented by phosphorylated ATM and Rad51. The status of proton therapy energy used to treat major types of cancer is summarized. Here, human lymphocytes from eight healthy donors (male and female) were irradiated with a spread-out Bragg peak using a therapeutic 70 MeV proton beam or with reference X rays. For both types of radiation, the kinetics of pATM and Rad51 repair protein activation (0–24 h) were estimated as determinants of homologous and non-homologous double-strand break repair. Additionally, γ-H2AX was used as the gold standard marker of double-strand breaks. Our results showed that at 30 min postirradiation there was significantly greater accumulation of γ-H2AX (0.6-fold), pATM (2.0-fold), and Rad51 (0.6-fold) in the proton-irradiated cells compared with the X-ray-treated cells. At 24 h post irradiation, for both types of radiation and all investigated proteins, the foci number was still significantly higher when compared with control. Furthermore, the mean value of pATM and Rad51 repair effectiveness was higher in cells exposed to protons than in cells exposed to X rays; however, the difference was significant only for pATM. The largest inter-individual differences in the repair capabilities were noted for Rad51. The association between the frequency of repair protein foci and the frequency of lymphocyte viability at 1 h post irradiation showed a positive correlation for protons but a negative correlation for X rays. These findings indicate that the accumulation of radiation-induced repair protein foci after proton versus X-ray irradiation differs between patients, consequently affecting the cellular responses to particle therapy and conventional radiation therapy.

Rapid genome editing by CRISPR-Cas9-POLD3 fusion

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein - Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.

Structure of HIV-1 Vpr in complex with the human nucleotide excision repair protein hHR23A

HIV-1 Vpr is a prototypic member of a large family of structurally related lentiviral virulence factors that antagonize various aspects of innate antiviral immunity. It subverts host cell DNA repair and protein degradation machineries by binding and inhibiting specific post-replication repair enzymes, linking them via the DCAF1 substrate adaptor to the Cullin 4 RING E3 ligase (CRL4DCAF1). HIV-1 Vpr also binds to the multi-domain protein hHR23A, which interacts with the nucleotide excision repair protein XPC and shuttles ubiquitinated proteins to the proteasome. Here, we report the atomic resolution structure of Vpr in complex with the C-terminal half of hHR23A, containing the XPC-binding (XPCB) and ubiquitin-associated (UBA2) domains. The XPCB and UBA2 domains bind to different sides of Vpr’s 3-helix-bundle structure, with UBA2 interacting with the α2 and α3 helices of Vpr, while the XPCB domain contacts the opposite side of Vpr’s α3 helix. The structure as well as biochemical results reveal that hHR23A and DCAF1 use overlapping binding surfaces on Vpr, even though the two proteins exhibit entirely different three-dimensional structures. Our findings show that Vpr independently targets hHR23A- and DCAF1- dependent pathways and highlight HIV-1 Vpr as a versatile module that interferes with DNA repair and protein degradation pathways.