Artificial pathway emergence in central metabolism from three recursive phosphoketolase reactions.

The promiscuous activities of a recursive, generalist enzyme provide raw material for the emergence of metabolic pathways. Here, we use a synthetic biology approach to recreate such an evolutionary setup in central metabolism and explore how cellular physiology adjusts to enable recursive catalysis. We generate an Escherichia coli strain deleted in transketolase and glucose 6-phosphate dehydrogenase, effectively eliminating the native pentose phosphate pathway. We demonstrate that the overexpression of phosphoketolase restores prototrophic growth by catalyzing three consecutive reactions, cleaving xylulose 5-phosphate, fructose 6-phosphate, and, notably, sedoheptulose 7-phosphate. We find that the activity of the resulting synthetic pathway becomes possible due to the recalibration of steady-state concentrations of key metabolites, such that the in vivo cleavage rates of all three phosphoketolase substrates are similar. This study demonstrates our ability to rewrite one of nature's most conserved pathways and provides insight into the flexibility of cellular metabolism during pathway emergence.

More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny.

The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.

DNA polymerase µ is a global player in the repair of non-homologous end-joining substrates.

The specialized DNA polymerase µ (pol µ) intervenes in the repair mechanism non-homologous end-joining (NHEJ) as an end-processing factor but its role has not been fully elucidated. Pol µ has been shown to participate in DNA synthesis at junctions in vitro, including on unpaired substrates, and to promote annealing. However, the phenotypes observed in vivo poorly recapitulate the functions of pol µ reported in vitro. We analysed the repair of DNA double-strand breaks (DSBs) in a cellular context using improved NHEJ substrates. These substrates do not replicate in mammalian cells, thereby result in clonal repair events, which allows the measure of the efficiency of repair. We validated this paradigm by comparing the repair of NHEJ substrates to the repair reported for chromosome DSBs in mouse cells. Molecular analysis and, in most cases sequencing of more than 1500 repair events on a variety of NHEJ substrates in wild type and pol µ(-/-) mouse embryonic fibroblasts shows that, unexpectedly, the absence of pol µ decreases the efficiency of joining of all types of DSBs, including those that do not undergo end-processing. Importantly, by reducing the efficiency of accurate events, lack of pol µ also affects the overall fidelity of the repair process. We also show that, although pol µ does not help protect DNA ends from resection, the efficiency of repair of resected ends is reduced in the absence of pol µ. Interestingly, the DNA synthesis activity of pol µ, including on non-aligned substrates, appears negligible at least in a cellular context. Our data point to a critical role for pol µ as a global repair player that increases the efficiency and the fidelity of NHEJ.

Lack of DNA polymerase µ affects the kinetics of DNA double-strand break repair and impacts on cellular senescence.

The specialised DNA polymerase µ (pol µ) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo. These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol µ intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol µâ»(/)⁻ mouse embryonic fibroblasts (MEFs) we show that lack of pol µ results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol µ. Here we show pronounced senescence in pol µâ»(/)⁻ MEFs, associated with high levels of the tumor-suppressor p16(INK4A) and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol µâ»(/)⁻ MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol µ. Pol µâ»(/)⁻ MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate.

An end-joining repair mechanism in Escherichia coli.

Bridging broken DNA ends via nonhomologous end-joining (NHEJ) contributes to the evolution and stability of eukaryote genomes. Although some bacteria possess a simplified NHEJ mechanism, the human commensal Escherichia coli is thought to rely exclusively on homology-directed mechanisms to repair DNA double-strand breaks (DSBs). We show here that laboratory and pathogenic E. coli strains possess a distinct end-joining activity that repairs DSBs and generates genome rearrangements. This mechanism, named alternative end-joining (A-EJ), does not rely on the key NHEJ proteins Ku and Ligase-D which are absent in E. coli. Differently from classical NHEJ, A-EJ is characterized by extensive end-resection largely due to RecBCD, by overwhelming usage of microhomology and extremely rare DNA synthesis. We also show that A-EJ is dependent on the essential Ligase-A and independent on Ligase-B. Importantly, mutagenic repair requires a functional Ligase-A. Although generally mutagenic, accurate A-EJ also occurs and is frequent in some pathogenic bacteria. Furthermore, we show the acquisition of an antibiotic-resistance gene via A-EJ, refuting the notion that bacteria gain exogenous sequences only by recombination-dependent mechanisms. This finding demonstrates that E. coli can integrate unrelated, nonhomologous exogenous sequences by end-joining and it provides an alternative strategy for horizontal gene transfer in the bacterial genome. Thus, A-EJ contributes to bacterial genome evolution and adaptation to environmental challenges. Interestingly, the key features of A-EJ also appear in A-NHEJ, an alternative end-joining mechanism implicated in chromosomal translocations associated with human malignancies, and we propose that this mutagenic repair might have originated in bacteria.