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Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at the Thr2609 cluster is essential for its complete function in DNA repair and tissue stem cell homeostasis. This phenomenon is demonstrated by congenital bone marrow failure occurring in DNA-PKcs(3A/3A) mutant mice, which require bone marrow transplantation (BMT) to prevent early mortality. Surprisingly, an increased incidence of spontaneous tumors, especially skin cancer, was observed in adult BMT-rescued DNA-PKcs(3A/3A) mice. Upon further investigation, we found that spontaneous γH2AX foci occurred in DNA-PKcs(3A/3A) skin biopsies and primary keratinocytes and that these foci overlapped with telomeres during mitosis, indicating impairment of telomere replication and maturation. Consistently, we observed significantly elevated frequencies of telomere fusion events in DNA-PKcs(3A/3A) cells as compared with wild-type and DNA-PKcs-knockout cells. In addition, a previously identified DNA-PKcs Thr2609Pro mutation, found in breast cancer, also induces a similar impairment of telomere leading-end maturation. Taken together, our current analyses indicate that the functional DNA-PKcs T2609 cluster is required to facilitate telomere leading strand maturation and prevention of genomic instability and cancer development.

DNA double-strand breaks (DSBs) are introduced in cells by ionizing radiation and reactive oxygen species. In addition, they are commonly generated during V(D)J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs: homologous recombination and non-homologous end-joining (NHEJ). The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.

WRN is unique among the five human RecQ DNA helicases in having a functional exonuclease domain (WRN-exo) and being defective in the premature aging and cancer-related disorder Werner syndrome. Here, we characterize WRN-exo crystal structures, biochemical activity and participation in DNA end joining. Metal-ion complex structures, active site mutations and activity assays reveal a nuclease mechanism mediated by two metal ions. The DNA end-binding Ku70/80 complex specifically stimulates WRN-exo activity, and structure-based mutational inactivation of WRN-exo alters DNA end joining in human cells. We furthermore establish structural and biochemical similarities of WRN-exo to DnaQ-family replicative proofreading exonucleases, describing WRN-specific adaptations consistent with double-stranded DNA specificity and functionally important conformational changes. These results indicate WRN-exo is a human DnaQ family member and support DnaQ-like proofreading activities stimulated by Ku70/80, with implications for WRN functions in age-related pathologies and maintenance of genomic integrity.

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24-/- mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24-/- MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.

Summary In the current study, we prospectively compared the diagnostic value of SW-CG-HYPR and conventional SRTurbo-FLASH for myocardial perfusion MRI in patients with suspected CAD. Compared with conventional SRTurbo-FLASH, SW-CG-HYPR allows increased spatial coverage (whole left ventricular coverage), improved temporal and spatial resolution and SNR, reduced motion artifacts and has higher diagnostic accuracy in patients with suspected CAD. Background A sliding-window (SW) conjugate-gradient (CG) highly constrained back-projection reconstruction (HYPR) (SW-CG-HYPR) technique has been developed for timeresolved myocardial perfusion imaging. Using this method, the acquisition time per cardiac cycle can be reduced dramatically while maintaining the temporal resolution of one frame per heartbeat in myocardial perfusion MRI, allowing increased spatial coverage (whole left ventricular coverage), improved temporal and spatial resolution and SNR, and reduced motion artifacts compared with the conventional SR-Turbo-FLASH sequence. However, the diagnostic accuracy of myocardial perfusion MRI with SW-CG-HYPR for detecting coronary artery disease (CAD) has not been directly compared to that with conventional SR-Turbo-FLASH. The purpose of this study was to prospectively compare the diagnostic value of SW-CG-HYPR and conventional SR-Turbo-FLASH for myocardial perfusion MRI in patients with suspected CAD. Methods

In this work, we have prospectively examined the diagnostic value of adenosine-induced stress myocardial perfusion MRI using SW-CG-HYPR in 50 patients with suspected CAD. Using the high-resolution, whole left ventricle SW-CG-HYPR method, perfusion MRI was able to depict hemodynamically relevant coronary artery stenosis with an accuracy of 90% and 93% based on per-patient and per-vessel analyses, respectively, using x-ray coronary angiography as the reference standard.

A mammalian apurinic/apyrimidinic endonuclease (AP endonuclease) is known to have two distinct functional domains. One domain is responsible for regulating the activity of Fos/Jun proto-oncogene products to bind to DNA at specific recognition sites. The other domain, which is highly conserved from bacteria to mammals, is responsible for repairing DNA damage caused by ionizing radiation, oxidative damage, and alkylating agents. This study reports on the isolation and characterization of the genomic structure of the mouse AP endonuclease gene (Apex). The genomic sequence of the Apex gene was 2.14 kb in length and contained four exons. Exon 1 contained a 0.24-kb untranslated 5' region upstream of the initiation codon. Consensus sequences for two CAAT boxes and a GC box were found upstream of the end of exon 1. A polymorphism was noted in the untranslated region of exon 1 in a comparison of a number of mouse strains. These data indicate that the 5' end of the mouse gene (Apex) differs from the previously isolated human gene (Ape), which has five exons and an untranslated region between exons 1 and 2. Data are also presented that suggest the presence of two pseudogenes in the mouse.

A systematic method for automatic layout synthesis of analog integrated circuit modules is presented. This method uses analog circuit recognition and critical net analysis techniques to derive proper layout constraints for analog circuit performance optimization. These layout constraints are analyzed and prioritized according to the recognized analog circuit topologies and classified net sensitivities. The weighted constraints are then used to drive the physical layout generation process to obtain a high-quality custom circuit layout. An efficient, constraint-driven analog floorplanning technique based on a zone-sensitivity partitioning algorithm is specially developed to generate a slicing floorplan incorporating the layout constraints. This layout synthesis approach has three key advantages. First, it can produce a satisfactory analog circuit performance with negligible degradation due to the layout-introduced parasitic effects. Second, it allows a complete automation for netlist-to-layout synthesis so that the layout tool can be used by VLSI system designers. Finally, this method is quite general and can be applied to handle a wide variety of analog circuits. Experimental results in CMOS operational amplifiers and a comparator are presented.

Microarray analysis of the DNA-dependent protein kinase catalytic subunit knock-out mouse: global effects on gene regulation and the cellular response to ionizing radiation