High-Throughput Analysis of Heteroduplex DNA in Mitotic Recombination Products.

Mitotic double-strand breaks (DSBs) are repaired by recombination with a homologous donor duplex. This process involves the exchange of single DNA strands between the broken molecule and the repair template, giving rise to regions of heteroduplex DNA (hetDNA). The creation of a defined DSB coupled with the use of a sequence-diverged repair template allows the fine-structure mapping of hetDNA through the sequencing of recombination products. A high-throughput method is described that capitalizes on the single-molecule real-time (SMRT) sequencing technology developed by PacBio. This method allows simultaneous analysis of the hetDNA contained within hundreds of recombination products.

Recombinational Repair of Nuclease-Generated Mitotic Double-Strand Breaks with Different End Structures in Yeast.

Mitotic recombination is the predominant mechanism for repairing double-strand breaks in Saccharomyces cerevisiae Current recombination models are largely based on studies utilizing the enzyme I-SceI or HO to create a site-specific break, each of which generates broken ends with 3' overhangs. In this study sequence-diverged ectopic substrates were used to assess whether the frequent Pol δ-mediated removal of a mismatch 8 nucleotides from a 3' end affects recombination outcomes and whether the presence of a 3' vs. 5' overhang at the break site alters outcomes. Recombination outcomes monitored were the distributions of recombination products into crossovers vs. noncrossovers, and the position/length of transferred sequence (heteroduplex DNA) in noncrossover products. A terminal mismatch that was 22 nucleotides from the 3' end was rarely removed and the greater distance from the end did not affect recombination outcomes. To determine whether the recombinational repair of breaks with 3' vs. 5' overhangs differs, we compared the well-studied 3' overhang created by I-SceI to a 5' overhang created by a ZFN (Zinc Finger Nuclease). Initiation with the ZFN yielded more recombinants, consistent with more efficient cleavage and potentially faster repair rate relative to I-SceI. While there were proportionally more COs among ZFN- than I-SceI-initiated events, NCOs in the two systems were indistinguishable in terms of the extent of strand transfer. These data demonstrate that the method of DSB induction and the resulting differences in end polarity have little effect on mitotic recombination outcomes despite potential differences in repair rate.

Glucose 6-phosphate dehydrogenase deficiency increases redox stress and moderately accelerates the development of heart failure.

BACKGROUND: Glucose 6-phosphate dehydrogenase (G6PD) is the most common deficient enzyme in the world. In failing hearts, G6PD is upregulated and generates reduced nicotinamide adenine dinucleotide phosphate (NADPH) that is used by the glutathione pathway to remove reactive oxygen species but also as a substrate by reactive oxygen species-generating enzymes. Therefore, G6PD deficiency might prevent heart failure by decreasing NADPH and reactive oxygen species production. METHODS AND RESULTS: This hypothesis was evaluated in a mouse model of human G6PD deficiency (G6PDX mice, ≈40% normal activity). Myocardial infarction with 3 months follow-up resulted in left ventricular dilation and dysfunction in both wild-type and G6PDX mice but significantly greater end diastolic volume and wall thinning in G6PDX mice. Similarly, pressure overload induced by transverse aortic constriction (TAC) for 6 weeks caused greater left ventricular dilation in G6PDX mice than wild-type mice. We further stressed transverse aortic constriction mice by feeding a high fructose diet to increase flux through G6PD and reactive oxygen species production and again observed worse left ventricular remodeling and a lower ejection fraction in G6PDX than wild-type mice. Tissue content of lipid peroxidation products was increased in G6PDX mice in response to infarction and aconitase activity was decreased with transverse aortic constriction, suggesting that G6PD deficiency increases myocardial oxidative stress and subsequent damage. CONCLUSIONS: Contrary to our hypothesis, G6PD deficiency increased redox stress in response to infarction or pressure overload. However, we found only a modest acceleration of left ventricular remodeling, suggesting that, in individuals with G6PD deficiency and concurrent hypertension or myocardial infarction, the risk for developing heart failure is higher but limited by compensatory mechanisms.