DNA-PKcs, ATM, and ATR Interplay Maintains Genome Integrity during Neurogenesis.

The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G2/M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system. SIGNIFICANCE STATEMENT: The DNA damage response (DDR) is essential for prevention of a broad spectrum of different human neurologic diseases. However, a detailed understanding of the DDR at a physiological level is lacking. In contrast to many in vitro cellular studies, here we demonstrate independent biological roles for the DDR kinases DNA-PKcs, ATM, and ATR during neurogenesis. We show that DNA-PKcs is central to DNA repair in nonproliferating cells, and restricts DNA damage accumulation, whereas ATR controls damage-induced G2 checkpoint control and apoptosis in proliferating cells. Conversely, ATM is critical for controlling apoptosis in immature noncycling neural cells after DNA damage. These data demonstrate functionally distinct, but cooperative, roles for each kinase in preserving genome stability in the nervous system.

ATR maintains select progenitors during nervous system development.

The ATR (ATM (ataxia telangiectasia mutated) and rad3-related) checkpoint kinase is considered critical for signalling DNA replication stress and its dysfunction can lead to the neurodevelopmental disorder, ATR-Seckel syndrome. To understand how ATR functions during neurogenesis, we conditionally deleted Atr broadly throughout the murine nervous system, or in a restricted manner in the dorsal telencephalon. Unexpectedly, in both scenarios, Atr loss impacted neurogenesis relatively late during neural development involving only certain progenitor populations. Whereas the Atr-deficient embryonic cerebellar external germinal layer underwent p53- (and p16(Ink4a/Arf))-independent proliferation arrest, other brain regions suffered apoptosis that was partially p53 dependent. In contrast to other organs, in the nervous system, p53 loss did not worsen the outcome of Atr inactivation. Coincident inactivation of Atm also did not affect the phenotype after Atr deletion, supporting non-overlapping physiological roles for these related DNA damage-response kinases in the brain. Rather than an essential general role in preventing replication stress, our data indicate that ATR functions to monitor genomic integrity in a selective spatiotemporal manner during neurogenesis.

Roles of nonhomologous end-joining pathways in surviving topoisomerase II-mediated DNA damage.

Topoisomerase II is a target for clinically active anticancer drugs. Drugs targeting these enzymes act by preventing the religation of enzyme-DNA covalent complexes leading to protein-DNA adducts that include single- and double-strand breaks. In mammalian cells, nonhomologous repair pathways are critical for repairing topoisomerase II-mediated DNA damage. Because topoisomerase II-targeting agents, such as etoposide, can also induce chromosomal translocations that can lead to secondary malignancies, understanding nonhomologous repair of topoisomerase II-mediated DNA damage may help to define strategies that limit this critical side effect on an important class of anticancer agents. Using Saccharomyces cerevisiae as a model eukaryote, we have determined the contribution of genes required for nonhomologous end-joining (NHEJ) for repairing DNA damage arising from treatment with topoisomerase II poisons, such as etoposide and 4'-(9-acridinylamino)methanesulfon-m-anisidide (mAMSA). To increase cellular sensitivity to topoisomerase II poisons, we overexpressed either wild-type or drug-hypersensitive alleles of yeast topoisomerase II. Using this approach, we found that yku70 (hdf1), yku80 (hdf2), and other genes required for NHEJ were important for cell survival following exposure to etoposide. The clearest increase in sensitivity was observed with cells overexpressing an etoposide-hypersensitive allele of TOP2 (Ser740Trp). Hypersensitivity was also seen in some end-joining defective mutants exposed to the intercalating agent mAMSA, although the increase in sensitivity was less pronounced. To confirm that the increase in sensitivity was not solely due to the elevated expression of TOP2 or due to specific effects of the drug-hypersensitive TOP2 alleles, we also found that deletion of genes required for NHEJ increased the sensitivity of rad52 deletions to both etoposide and mAMSA. Taken together, these results show a clear role for NHEJ in the repair of DNA damage induced by topoisomerase II-targeting agents and suggest that this pathway may participate in translocations generated by drugs, such as etoposide.

Nanopatterning proteins and peptides.

A variety of techniques have been developed to site-specifically immobilize biomolecules onto surfaces with resolutions below one micron. The ability to pattern proteins and peptides in particular has great potential for applications in biosensors, biomaterials, and tissue engineering. For example, immobilizing proteins at the nanoscale could lead to the development of diagnostic protein nanoarrays, while patterning peptides could lead to greater control over the cell/biomaterial interface. This review discusses the methods that have been reported for patterning proteins and peptides with submicron and nanometer resolutions.

Submicron streptavidin patterns for protein assembly.

Micron and submicron-scale features of aldehyde functionality were fabricated in polymer films by photolithography to develop a platform for protein immobilization and assembly at a biologically relevant scale. Films containing the pH-reactive polymer poly(3,3'-diethoxypropyl methacrylate) and a photoacid generator (PAG) were patterned from 500 nm to 40 mum by exposure to 365 nm (i-line) light. Upon PAG activation and hydrolysis of acetals, aldehyde groups formed. After the films were incubated with a biotinylated aldehyde reactive probe, the X-ray photoelectron spectroscopy results were consistent with biotin being attached to the surface. The background was subsequently passivated by flood exposure and incubation with an aminooxy-terminated poly(ethylene glycol), resulting in a 98% reduction in nonspecific protein adsorption. Protein patterning and assembly was demonstrated using streptavidin, biotinylated anthrax toxin receptor-1, and the protective antigen moiety of anthrax toxin and confirmed by fluorescence microscopy and atomic force microscopy (AFM). AFM demonstrated that 500 nm protein features were achieved. Because of the abundance of biotinylated proteins, this methodology provides a platform for protein immobilization and assembly for various applications in biotechnology.