Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP—labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells.DOI: http://dx.doi.org/10.7554/eLife.01222.001

Summary Actively dividing cells perform robust and accurate DNA replication during fluctuating nutrient availability, yet factors that prevent disruption of replication remain largely unknown. Here we report that DksA, a nutrient-responsive transcription factor, ensures replication completion in Escherichia coli by removing transcription roadblocks. In the absence of DksA, replication is rapidly arrested upon amino acid starvation. This arrest requires active transcription, and is alleviated by RNA polymerase mutants that compensate for DksA activity. This replication arrest occurs independently of exogenous DNA damage, yet it induces the DNA damage response and recruits the main recombination protein RecA. This novel function of DksA is independent of its transcription initiation activity, but requires its less studied transcription elongation activity. Finally, GreA/B elongation factors also prevent replication arrest during nutrient stress. We conclude that transcription elongation factors alleviate fundamental conflicts between replication and transcription, thereby protecting replication fork progression and DNA integrity.

There is mounting evidence that family functioning is linked to childhood overweight and obesity, and that both of these are associated with health-related behaviours and adverse health outcomes in children and adolescents. This paper systematically examines the peer-reviewed evidence regarding the relationship between child and adolescent overweight and obesity and family functioning. Peer-reviewed literature published between 1990 and 2011 hosted in Scopus, Pub Med or Psyc INFO were searched, in addition to the reference lists of included papers. Twenty-one studies met the selection criteria. Of the 17 identified cross-sectional and longitudinal studies, 12 reported significant associations between family functioning and childhood overweight and obesity. The instruments used to measure family functioning in the identified studies were heterogeneous. Poor family functioning was associated with increased risk of obesity and overweight in children and adolescents, and obese children and adolescents were more likely to come from families with poor family functioning. Aspects of family functioning which were associated with increased risk of child and adolescent obesity included poor communication, poor behaviour control, high levels of family conflict and low family hierarchy values. Half (2/4) of the identified intervention studies showed a significant relationship between family functioning and changes in child weight. The results demonstrate that family functioning is linked to obesity; however, higher level evidence and greater understanding of the mechanisms behind this relationship are required. The results indicate a need for a standardised family functioning measure applicable across populations. The results provide evidence of the value of considering family functioning in childhood obesity research and intervention.

Bistable epigenetic switches are fundamental for cell fate determination in unicellular and multicellular organisms. Regulatory proteins associated with bistable switches are often present in low numbers and subject to molecular noise. It is becoming clear that noise in gene expression can influence cell fate. Although the origins and consequences of noise have been studied, the stochastic and transient nature of RNA errors during transcription has not been considered in the origin or modeling of noise nor has the capacity for such transient errors in information transfer to generate heritable phenotypic change been discussed. We used a classic bistable memory module to monitor and capture transient RNA errors: the lac operon of Escherichia coli comprises an autocatalytic positive feedback loop producing a heritable all-or-none epigenetic switch that is sensitive to molecular noise. Using single-cell analysis, we show that the frequency of epigenetic switching from one expression state to the other is increased when the fidelity of RNA transcription is decreased due to error-prone RNA polymerases or to the absence of auxiliary RNA fidelity factors GreA and GreB (functional analogues of eukaryotic TFIIS). Therefore, transcription infidelity contributes to molecular noise and can effect heritable phenotypic change in genetically identical cells in the same environment. Whereas DNA errors allow genetic space to be explored, RNA errors may allow epigenetic or expression space to be sampled. Thus, RNA infidelity should also be considered in the heritable origin of altered or aberrant cell behaviour.