The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress.

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

MYSM1 attenuates DNA damage signals triggered by physiologic and genotoxic DNA breaks.

BACKGROUND: Patients with deleterious variants in MYSM1 have an immune deficiency characterized by B-cell lymphopenia, hypogammaglobulinemia, and increased radiosensitivity. MYSM1 is a histone deubiquitinase with established activity in regulating gene expression. MYSM1 also localizes to sites of DNA injury but its function in cellular responses to DNA breaks has not been elucidated. OBJECTIVES: This study sought to determine the activity of MYSM1 in regulating DNA damage responses (DDRs) to DNA double-stranded breaks (DSBs) generated during immunoglobulin receptor gene (Ig) recombination and by ionizing radiation. METHODS: MYSM1-deficient pre- and non-B cells were used to determine the role of MYSM1 in DSB generation, DSB repair, and termination of DDRs. RESULTS: Genetic testing in a newborn with abnormal screen for severe combined immune deficiency, T-cell lymphopenia, and near absence of B cells identified a novel splice variant in MYSM1 that results in nearly absent protein expression. Radiosensitivity testing in patient's peripheral blood lymphocytes showed constitutive γH2AX, a marker of DNA damage, in B cells in the absence of irradiation, suggesting a role for MYSM1 in response to DSBs generated during Ig recombination. Suppression of MYSM1 in pre-B cells did not alter generation or repair of Ig DSBs. Rather, loss of MYSM1 resulted in persistent DNA damage foci and prolonged DDR signaling. Loss of MYSM1 also led to protracted DDRs in U2OS cells with irradiation induced DSBs. CONCLUSIONS: MYSM1 regulates termination of DNA damage responses but does not function in DNA break generation and repair.

Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity.

The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin-containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A-induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.

The Zebrafish Xenograft Platform-A Novel Tool for Modeling KSHV-Associated Diseases.

Kaposi's sarcoma associated-herpesvirus (KSHV, also known as human herpesvirus-8) is a gammaherpesvirus that establishes life-long infection in human B lymphocytes. KSHV infection is typically asymptomatic, but immunosuppression can predispose KSHV-infected individuals to primary effusion lymphoma (PEL); a malignancy driven by aberrant proliferation of latently infected B lymphocytes, and supported by pro-inflammatory cytokines and angiogenic factors produced by cells that succumb to lytic viral replication. Here, we report the development of the first in vivo model for a virally induced lymphoma in zebrafish, whereby KSHV-infected PEL tumor cells engraft and proliferate in the yolk sac of zebrafish larvae. Using a PEL cell line engineered to produce the viral lytic switch protein RTA in the presence of doxycycline, we demonstrate drug-inducible reactivation from KSHV latency in vivo, which enabled real-time observation and evaluation of latent and lytic phases of KSHV infection. In addition, we developed a sensitive droplet digital PCR method to monitor latent and lytic viral gene expression and host cell gene expression in xenografts. The zebrafish yolk sac is not well vascularized, and by using fluorogenic assays, we confirmed that this site provides a hypoxic environment that may mimic the microenvironment of some human tumors. We found that PEL cell proliferation in xenografts was dependent on the host hypoxia-dependent translation initiation factor, eukaryotic initiation factor 4E2 (eIF4E2). This demonstrates that the zebrafish yolk sac is a functionally hypoxic environment, and xenografted cells must switch to dedicated hypoxic gene expression machinery to survive and proliferate. The establishment of the PEL xenograft model enables future studies that exploit the innate advantages of the zebrafish as a model for genetic and pharmacologic screens.

Textured insoles reduce vertical loading rate and increase subjective plantar sensation in overground running.

The effect of textured insoles on kinetics and kinematics of overground running was assessed. 16 male injury-free-recreational runners attended a single visit (age 23 ± 5 yrs; stature 1.78 ± 0.06 m; mass 72.6 ± 9.2 kg). Overground 15-m runs were completed in flat, canvas plimsolls both with and without textured insoles at self-selected velocity on an indoor track in an order that was balanced among participants. Average vertical loading rate and peak vertical force (Fpeak) were captured by force platforms. Video footage was digitised for sagittal plane hip, knee and ankle angles at foot strike and mid stance. Velocity, stride rate and length and contact and flight time were determined. Subjectively rated plantar sensation was recorded by visual scale. 95% confidence intervals estimated mean differences. Smallest worthwhile change in loading rate was defined as standardised reduction of 0.54 from a previous comparison of injured versus non-injured runners. Loading rate decreased (-25 to -9.3 BW s-1; 60% likely beneficial reduction) and plantar sensation was increased (46-58 mm) with the insole. Fpeak (-0.1 to 0.14 BW) and velocity (-0.02 to 0.06 m s-1) were similar. Stride length, flight and contact time were lower (-0.13 to -0.01 m; -0.02 to-0.01 s; -0.016 to -0.006 s) and stride rate was higher (0.01-0.07 steps s-1) with insoles. Textured insoles elicited an acute, meaningful decrease in vertical loading rate in short distance, overground running and were associated with subjectively increased plantar sensation. Reduced vertical loading rate could be explained by altered stride characteristics.

Movement of water infiltrated from a recharge basin to wells.

Local surface water and stormflow were infiltrated intermittently from a 40-ha basin between September 2003 and September 2007 to determine the feasibility of recharging alluvial aquifers pumped for public supply, near Stockton, California. Infiltration of water produced a pressure response that propagated through unconsolidated alluvial-fan deposits to 125 m below land surface (bls) in 5 d and through deeper, more consolidated alluvial deposits to 194 m bls in 25 d, resulting in increased water levels in nearby monitoring wells. The top of the saturated zone near the basin fluctuates seasonally from depths of about 15 to 20 m. Since the start of recharge, water infiltrated from the basin has reached depths as great as 165 m bls. On the basis of sulfur hexafluoride tracer test data, basin water moved downward through the saturated alluvial deposits until reaching more permeable zones about 110 m bls. Once reaching these permeable zones, water moved rapidly to nearby pumping wells at rates as high as 13 m/d. Flow to wells through highly permeable material was confirmed on the basis of flowmeter logging, and simulated numerically using a two-dimensional radial groundwater flow model. Arsenic concentrations increased slightly as a result of recharge from 2 to 6 µg/L immediately below the basin. Although few water-quality issues were identified during sample collection, high groundwater velocities and short travel times to nearby wells may have implications for groundwater management at this and at other sites in heterogeneous alluvial aquifers.