The DNA Damage Response: Making It Safe to Play with Knives

Autism is a heterogeneous syndrome defined by impairments in three core domains: social interaction, language and range of interests. Recent work has led to the identification of several autism susceptibility genes and an increased appreciation of the contribution of de novo and inherited copy number variation. Promising strategies are also being applied to identify common genetic risk variants. Systems biology approaches, including array-based expression profiling, are poised to provide additional insights into this group of disorders, in which heterogeneity, both genetic and phenotypic, is emerging as a dominant theme.

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Advances in autism genetics: on the threshold of a new neurobiology

The text consists of two sections, one for those studying or practicing diagnostic radiology, nuclear medicine and radiation oncology; the other for those engaged in the study or clinical practice of radiation oncology--a new chapter, on radiologic terrorism, is specifically for those in the radiation sciences who would manage exposed individuals in the event of a terrorist event. The 17 chapters in Section I represent a general introduction to radiation biology and a complete, self-contained course especially for residents in diagnostic radiology and nuclear medicine that follows the Syllabus in Radiation Biology of the RSNA. The 11 chapters in Section II address more in-depth topics in radiation oncology, such as cancer biology, retreatment after radiotherapy, chemotherapeutic agents and hyperthermia.

Radiobiology for the Radiologist

DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1(XRS2) complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.

/pdf/regulation-of-dna-double-strand-break-repair-pathway-choice-4nmgza2h52.pdf

Regulation of DNA double-strand break repair pathway choice

Recent findings have thrust poly(ADP-ribose) polymerases (PARPs) into the limelight as potential chemotherapeutic targets. To provide a framework for understanding these recent observations, we review what is known about the structures and functions of the family of PARP enzymes, and then outline a series of questions that should be addressed to guide the rational development of PARP inhibitors as anticancer agents.

PARP inhibition: PARP1 and beyond

Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer.

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

Biochemical and genetic studies support the view that the majority of DNA double-strand breaks induced in the genome of higher eukaryotes by ionizing radiation are removed by two pathways of nonhomologous end joining (NHEJ) termed D-NHEJ and B-NHEJ. Whereas D-NHEJ depends on the activities of the DNA-dependent protein kinase and DNA ligase IV/XRCC4, components of B-NHEJ have not been identified. Using extract fractionation, we show that the majority of DNA end joining activity in extracts of HeLa cells derives from DNA ligase III. DNA ligase III fractionates through two columns with the maximum in DNA end joining activity and its depletion from the extract causes loss of activity that can be recovered by the addition of purified enzyme. The same fractionation protocols provide evidence for an additional factor strongly enhancing DNA end joining and shifting the product spectrum from circles to multimers. An in vivo plasmid assay shows that DNA ligase IV–deficient mouse embryo fibroblasts retain significant DNA end joining activity that can be reduced by up to 80% by knocking down DNA ligase III using RNA interference. These in vivo and in vitro observations identify DNA ligase III as a candidate component for B-NHEJ and point to additional factors contributing to NHEJ efficiency.

https://cancerres.aacrjournals.org/content/canres/65/10/4020.full.pdf

DNA Ligase III as a Candidate Component of Backup Pathways of Nonhomologous End Joining

Central nervous system (CNS) risks which include during space missions and lifetime risks due to space radiation exposure are of concern for long-term exploration missions to Mars or other destinations. Possible CNS risks during a mission are altered cognitive function, including detriments in short-term memory, reduced motor function, and behavioral changes, which may affect performance and human health. The late CNS risks are possible neurological disorders such as premature aging, and Alzheimer's disease (AD) or other dementia. Radiation safety requirements are intended to prevent all clinically significant acute risks. However the definition of clinically significant CNS risks and their dependences on dose, dose-rate and radiation quality is poorly understood at this time. For late CNS effects such as increased risk of AD, the occurrence of the disease is fatal with mean time from diagnosis of early stage AD to death about 8 years. Therefore if AD risk or other late CNS risks from space radiation occur at mission relevant doses, they would naturally be included in the overall acceptable risk of exposure induced death (REID) probability for space missions. Important progress has been made in understanding CNS risks due to space radiation exposure, however in general the doses used in experimental studies have been much higher than the annual galactic cosmic ray (GCR) dose (∼0.1 Gy/y at solar maximum and ∼0.2 Gy/y at solar minimum with less than 50% from HZE particles). In this report we summarize recent space radiobiology studies of CNS effects from particle accelerators simulating space radiation using experimental models, and make a critical assessment of their relevance relative to doses and dose-rates to be incurred on a Mars mission. Prospects for understanding dose, dose-rate and radiation quality dependencies of CNS effects and extrapolation to human risk assessments are described.

Space radiation risks to the central nervous system

In eukaryotic cells, DNA double strand breaks (DSBs) cause the prompt phosphorylation of serine 139 at the carboxy terminus of histone H2AX to generate gamma-H2AX, detectable by Western blotting or immunofluorescence. The consensus sequence at the phosphorylation site implicates the phosphatidylinositol 3-like family of protein kinases in H2AX phosphorylation. It remains open whether ATM (ataxia telangiectasia mutated) is the major H2AX kinase, or whether other members of the family, such as DNA-PK (DNA dependent protein kinase) or ATR (ATM and Rad3 related), contribute in a functionally complementary manner. To address this question, we measured global H2AX phosphorylation in cell lysates and foci formation in individual cells of either wild type or mutant (ATM or DNA-PK) genetic background. Normal global phosphorylation kinetics is observed after irradiation in cells defective either in ATM or DNA-PK alone, suggesting a complementary contribution to H2AX phosphorylation. This is further supported by the observation that initial H2AX phosphorylation is delayed when both kinases are inhibited by wortmannin, as well as when ATM is inhibited by caffeine in DNA-PK deficient cells. However, robust residual global phosphorylation is detectable under all conditions of genetic or chemical inhibition suggesting the function of additional kinases, such as ATR. Treatment with wortmannin, caffeine, or UCN-01 produces a strong DNA-PK dependent late global hyperphosphorylation of H2AX, uncoupled from DNA DSB rejoining and compatible with an inhibition of late steps in DNA DSB processing. Evaluation of gamma-H2AX foci formation confirms the major conclusions made on the basis of global H2AX phosphorylation, but also points to differences particularly several hours after exposure to IR. The results in aggregate implicate DNA-PK, ATM and possibly other kinases in H2AX phosphorylation. The functional significance and the mechanisms of coordination in space and time of these multiple inputs require further investigation.

Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors

In cells of higher eukaryotes double strand breaks (DSBs) induced in the DNA after exposure to ionizing radiation (IR) are rapidly rejoined by a pathway of non-homologous end joining (NHEJ) that requires DNA dependent protein kinase (DNA-PK) and is therefore termed here D-NHEJ. When this pathway is chemically or genetically inactivated, cells still remove the majority of DSBs using an alternative, backup pathway operating independently of the RAD52 epistasis group of genes and with an order of magnitude slower kinetics (B-NHEJ). Here, we investigate the role of DNA-PK in the functional coordination of D-NHEJ and B-NHEJ using as a model end joining by cell extracts of restriction endonuclease linearized plasmid DNA. Although DNA end joining is inhibited by wortmannin, an inhibitor of DNA-PK, the degree of inhibition depends on the ratio between DNA ends and DNA-PK, suggesting that binding of inactive DNA-PK to DNA ends not only blocks processing by D-NHEJ, but also prevents the function of B-NHEJ. Residual end joining under conditions of incomplete inhibition, or in cells lacking DNA-PK, is attributed to the function of B-NHEJ operating on DNA ends free of DNA-PK. Thus, DNA-PK suppresses alternative pathways of end joining by efficiently binding DNA ends and shunting them to D-NHEJ.

Minli Wang

Papers

PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

DNA Ligase III as a Candidate Component of Backup Pathways of Nonhomologous End Joining

Space radiation risks to the central nervous system

Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors

Backup pathways of NHEJ are suppressed by DNA-PK.