Failure to repair endogenous DNA damage in ß-cells causes adult-onset diabetes in mice.

Age is the greatest risk factor for the development of type 2 diabetes mellitus (T2DM). Age-related decline in organ function is attributed to the accumulation of stochastic damage, including damage to the nuclear genome. Islets of T2DM patients display increased levels of DNA damage. However, whether this is a cause or consequence of the disease has not been elucidated. Here, we asked if spontaneous, endogenous DNA damage in ß-cells can drive ß-cell dysfunction and diabetes, via deletion of Ercc1, a key DNA repair gene, in ß-cells. Mice harboring Ercc1-deficient ß-cells developed adult-onset diabetes as demonstrated by increased random and fasted blood glucose levels, impaired glucose tolerance, and reduced insulin secretion. The inability to repair endogenous DNA damage led to an increase in oxidative DNA damage and apoptosis in ß-cells and a significant loss of ß-cell mass. Using electron microscopy, we identified ß-cells in clear distress that showed an increased cell size, enlarged nuclear size, reduced number of mature insulin granules, and decreased number of mitochondria. Some ß-cells were more affected than others consistent with the stochastic nature of spontaneous DNA damage. Ercc1-deficiency in ß-cells also resulted in loss of ß-cell function as glucose-stimulated insulin secretion and mitochondrial function were impaired in islets isolated from mice harboring Ercc1-deficient ß-cells. These data reveal that unrepaired endogenous DNA damage is sufficient to drive ß-cell dysfunction and provide a mechanism by which age increases the risk of T2DM.

Low Affinity DnaA-ATP Recognition Sites in E. coli oriC Make Non-equivalent and Growth Rate-Dependent Contributions to the Regulated Timing of Chromosome Replication.

Although the mechanisms that precisely time initiation of chromosome replication in bacteria remain unclear, most clock models are based on accumulation of the active initiator protein, DnaA-ATP. During each cell division cycle, sufficient DnaA-ATP must become available to interact with a distinct set of low affinity recognition sites in the unique chromosomal replication origin, oriC, and assemble the pre-replicative complex (orisome) that unwinds origin DNA and helps load the replicative helicase. The low affinity oriC-DnaA-ATP interactions are required for the orisome's mechanical functions, and may also play a role in timing of new rounds of DNA synthesis. To further examine this possibility, we constructed chromosomal oriCs with equal preference for DnaA-ADP or DnaA-ATP at one or more low affinity recognition sites, thereby lowering the DnaA-ATP requirement for orisome assembly, and measured the effect of the mutations on cell cycle timing of DNA synthesis. Under slow growth conditions, mutation of any one of the six low affinity DnaA-ATP sites in chromosomal oriC resulted in initiation earlier in the cell cycle, but the shift was not equivalent for every recognition site. Mutation of τ2 caused a greater change in initiation age, suggesting its occupation by DnaA-ATP is a temporal bottleneck during orisome assembly. In contrast, during rapid growth, all origins with a single mutated site displayed wild-type initiation timing. Based on these observations, we propose that E. coli uses two different, DnaA-ATP-dependent initiation timing mechanisms; a slow growth timer that is directly coupled to individual site occupation, and a fast growth timer comprising DnaA-ATP and additional factors that regulate DnaA access to oriC. Analysis of origins with paired mutated sites suggests that Fis is an important component of the fast growth timing mechanism.

Origin recognition is the predominant role for DnaA-ATP in initiation of chromosome replication.

In all cells, initiation of chromosome replication depends on the activity of AAA+ initiator proteins that form complexes with replication origin DNA. In bacteria, the conserved, adenosine triphosphate (ATP)-regulated initiator protein, DnaA, forms a complex with the origin, oriC, that mediates DNA strand separation and recruitment of replication machinery. Complex assembly and origin activation requires DnaA-ATP, which differs from DnaA-ADP in its ability to cooperatively bind specific low affinity sites and also to oligomerize into helical filaments. The degree to which each of these activities contributes to the DnaA-ATP requirement for initiation is not known. In this study, we compared the DnaA-ATP dependence of initiation from wild-type Escherichia coli oriC and a synthetic origin (oriCallADP), whose multiple low affinity DnaA sites bind DnaA-ATP and DnaA-ADP similarly. OriCallADP was fully occupied and unwound by DnaA-ADP in vitro, and, in vivo, oriCallADP suppressed lethality of DnaA mutants defective in ATP binding and ATP-specific oligomerization. However, loss of preferential DnaA-ATP binding caused over-initiation and increased sensitivity to replicative stress. The findings indicate both DnaA-ATP and DnaA-ADP can perform most of the mechanical functions needed for origin activation, and suggest that a key reason for ATP-regulation of DnaA is to control replication initiation frequency.

Spontaneous DNA damage to the nuclear genome promotes senescence, redox imbalance and aging.

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5-131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.

Safety and Outcome Measures of First-in-Human Intraperitoneal α Radioimmunotherapy With 212Pb-TCMC-Trastuzumab.

PURPOSE: One-year monitoring of patients receiving intraperitoneal (IP) Pb-TCMC-trastuzumab to provide long-term safety and outcome data. A secondary objective was to study 7 tumor markers for correlation with outcome. METHODS: Eighteen patients with relapsed intra-abdominal human epidermal growth factor receptor-2 expressing peritoneal metastases were treated with a single IP infusion of Pb-TCMC-trastuzumab, delivered <4 h after 4 mg/kg IV trastuzumab. Seven tumor markers were studied for correlation with outcome. RESULTS: Six dose levels (7.4, 9.6, 12.6, 16.3, 21.1, 27.4 MBq/m) were well tolerated with early possibly agent-related adverse events being mild, transient, and not dose dependent. These included asymptomatic, abnormal laboratory values. No late renal, liver, cardiac, or other toxicity was noted up to 1 year. There were no clinical signs or symptoms of an immune response to Pb-TCMC-trastuzumab, and assays to detect an immune response to this conjugate were negative for all tested. Tumor marker studies in ovarian cancer patients showed a trend of decreasing Cancer antigen 72-4 (CA 72-4) aka tumor-associated glycoprotein 72 (TAG-72) and tumor growth with increasing administered radioactivity. Other tumor markers, including carbohydrate antigen (CA125), human epididymis protein 4 (HE-4), serum amyloid A (SAA), mesothelin, interleukin-6 (IL-6), and carcinoembryonic antigen (CEA) did not correlate with imaging outcome. CONCLUSIONS: IP Pb-TCMC-trastuzumab up to 27 MBq/m seems safe for patients with peritoneal carcinomatosis who have failed standard therapies. Serum TAG-72 levels better correlated to imaging changes in ovarian cancer patients than the more common tumor marker, CA125.

Building the bacterial orisome: high-affinity DnaA recognition plays a role in setting the conformation of oriC DNA.

During assembly of the E. coli pre-replicative complex (pre-RC), initiator DnaA oligomers are nucleated from three widely separated high-affinity DnaA recognition sites in oriC. Oligomer assembly is then guided by low-affinity DnaA recognition sites, but is also regulated by a switch-like conformational change in oriC mediated by sequential binding of two DNA bending proteins, Fis and IHF, serving as inhibitor and activator respectively. Although their recognition sites are separated by up to 90 bp, Fis represses IHF binding and weak DnaA interactions until accumulating DnaA displaces Fis from oriC. It remains unclear whether high-affinity DnaA binding plays any role in Fis repression at a distance and it is also not known whether all high-affinity DnaA recognition sites play an equivalent role in oligomer formation. To examine these issues, we developed origin-selective recombineering methods to mutate E. coli chromosomal oriC. We found that, although oligomers were assembled in the absence of any individual high-affinity DnaA binding site, loss of DnaA binding at peripheral sites eliminated Fis repression, and made binding of both Fis and IHF essential. We propose a model in which interaction of DnaA molecules at high-affinity sites regulates oriC DNA conformation.

DNA damage triggers a chronic autoinflammatory response, leading to fat depletion in NER progeria.

Lipodystrophies represent a group of heterogeneous disorders characterized by loss of fat tissue. However, the underlying mechanisms remain poorly understood. Using mice carrying an ERCC1-XPF DNA repair defect systematically or in adipocytes, we show that DNA damage signaling triggers a chronic autoinflammatory response leading to fat depletion. Ercc1-/- and aP2-Ercc1F/- fat depots show extensive gene expression similarities to lipodystrophic Pparγ(ldi/+) animals, focal areas of ruptured basement membrane, the reappearance of primary cilia, necrosis, fibrosis, and a marked decrease in adiposity. We find that persistent DNA damage in aP2-Ercc1F/- fat depots and in adipocytes ex vivo triggers the induction of proinflammatory factors by promoting transcriptionally active histone marks and the dissociation of nuclear receptor corepressor complexes from promoters; the response is cell autonomous and requires ataxia telangiectasia mutated (ATM). Thus, persistent DNA damage-driven autoinflammation plays a causative role in adipose tissue degeneration, with important ramifications for progressive lipodystrophies and natural aging.

Two oppositely oriented arrays of low-affinity recognition sites in oriC guide progressive binding of DnaA during Escherichia coli pre-RC assembly.

The onset of chromosomal DNA replication requires highly precise and reproducible interactions between initiator proteins and replication origins to assemble a pre-replicative complex (pre-RC) that unwinds the DNA duplex. In bacteria, initiator protein DnaA, bound to specific high- and low-affinity recognition sites within the unique oriC locus, comprises the pre-RC, but how complex assembly is choreographed to ensure precise initiation timing during the cell cycle is not well understood. In this study, we present evidence that higher-order DnaA structures are formed at oriC when DnaA monomers are closely positioned on the same face of the DNA helix by interaction with two oppositely oriented essential arrays of closely spaced low-affinity DnaA binding sites. As DnaA levels increase, peripheral high-affinity anchor sites begin cooperative loading of the arrays, which is extended by sequential binding of additional DnaA monomers resulting in growth of the complexes towards the centre of oriC. We suggest that this polarized assembly of unique DnaA oligomers within oriC plays an important role in mediating pre-RC activity and may be a feature found in all bacterial replication origins.