DNA Double Strand Break Repair and Its Control by Nucleosome Remodeling

DNA double strand breaks (DSBs) are repaired in eukaryotes by one of several cellular mechanisms. The decision-making process controlling DSB repair takes place at the step of DNA end resection, the nucleolytic processing of DNA ends, which generates single-stranded DNA overhangs. Dependent on the length of the overhang, a corresponding DSB repair mechanism is engaged. Interestingly, nucleosomes—the fundamental unit of chromatin—influence the activity of resection nucleases and nucleosome remodelers have emerged as key regulators of DSB repair. Nucleosome remodelers share a common enzymatic mechanism, but for global genome organization specific remodelers have been shown to exert distinct activities. Specifically, different remodelers have been found to slide and evict, position or edit nucleosomes. It is an open question whether the same remodelers exert the same function also in the context of DSBs. Here, we will review recent advances in our understanding of nucleosome remodelers at DSBs: to what extent nucleosome sliding, eviction, positioning and editing can be observed at DSBs and how these activities affect the DSB repair decision.

Investigation of blaKPC and blaNDM genes in enterobacteriaceae received in a public health laboratory

Introduction: KPC and NDM carbapenemases production is an important enzymatic mechanism of resistance to carbapenens in bacteria belonging to the Enterobacteriaceae family. These enzymes degrade virtually all beta-lactam antibiotics and are encoded by the blaKPC and blaNDM genes, which can be in mobile genetic elements such as plasmids and transposons. Objectives: This study evaluated the positivity rate of the presence of blaKPC and blaNDM genes in carbapenem-resistant enterobacteria received at the Instituto Adolfo Lutz (IAL) of São José do Rio Preto, Brazil and determined the epidemiological data related to the patients whose isolates were recovered. Methods: From June 2015 to April 2019, bacterial isolates were obtained from different hospitals located in five municipalities in São José do Rio Preto region. In the bacteriology and molecular biology laboratory, DNA extraction and real-time PCR were performed to investigate the blaKPC and blaNDM genes. Afterwards, epidemiological data were surveyed such as the municipality of origin, age, and gender of the patients whose bacterial isolates were recovered. Results: A total of 934 enterobacteria isolates were recovered from the different hospitals. Of these; 93.4% were positive for blaKPC, with 96.3%, 1.85%, and 1.85% of the isolates belonged to the Klebsiella genus, Enterobacter genus, and Escherichia coli species, respectively. Also, 52.5% and 84.4% of the isolates were obtained from women and elderly patients, respectively. The blaNDM gene was detected only in three isolates, two of which originated from surveillance cultures. Conclusion: Therefore, KPC-producing enterobacteria are widespread in all health units of the five municipalities that were studied, suggesting that the blaKPC-carrying Klebsiella sp. isolates may be endemic in these institutions. Additionally, there is a significant role of surveillance cultures in preventing the spread of resistance genes, as observed for blaNDM in this study.

Oleate Hydratase in Lactobacillus delbrueckii subsp. bulgaricus LBP UFSC 2230 Catalyzes the Reversible Conversion between Linoleic Acid and Ricinoleic Acid

This study provides insight into the enzymatic mechanism of CLA synthesis in L. delbrueckii subsp. bulgaricus and broadens our understanding of the bioconversion of LA and RA by OleH. The impact of OleH on the production of the c 9, t 11 CLA isomer and stress tolerance by E. coli has been assisted.

Structure of glycine N-acyltransferase clarifies its catalytic mechanism

Glycine N-acyltransferase (GLYAT; EC 2.3.1.13) is a key enzyme in mammalian homeostasis that has been linked to several pathologies in humans, including cancer. Here we report the first crystal structure of a member of the GLYAT family, and a detailed interpretation of its structure and enzymatic mechanism. This work will aid further studies on GLYAT and its involvement in metabolic diseases and cancer.

Ligase A and RNase HI Participate in Completing Replication on the Chromosome in Escherichia coli

In Escherichia coli, several enzymes have been identified that participate in completing replication on the chromosome, including RecG, SbcCD, ExoI, and RecBCD. However, other enzymes are likely to be involved and the precise enzymatic mechanism by which this reaction occurs remains unknown. Two steps predicted to be necessary to complete replication are removal of Okazaki RNA fragments and ligation of the nascent strands at convergent replication forks. E. coli encodes two RNases that remove RNA-DNA hybrids, rnhA and rnhB, as well as two ligases, ligA and ligB. Here, we used replication profiling to show that rnhA and ligA, encoding RNase HI and Ligase A, participate in the completion reaction. Deletion of rnhA impaired the ability to complete replication and resulted in over-replication in the terminus region. It additionally suppressed initiation events from oriC, suggesting a role for the enzyme in oriC-dependent initiation, as has been suggested previously. We also show that a temperature-sensitive mutation in Ligase A led to over-replication at sites where replication completes, and that degradation at these sites occurred upon shifting to the nonpermissive temperature. Deletion of rnhB or ligB did not affect the growth or profile of replication on the genome.

Inhibitors of Fumarylacetoacetate Hydrolase Domain Containing Protein 1 (FAHD1)

FAH domain containing protein 1 (FAHD1) acts as oxaloacetate decarboxylase in mitochondria, contributing to the regulation of the tricarboxylic acid cycle. Guided by a high-resolution X-ray structure of FAHD1 liganded by oxalate, the enzymatic mechanism of substrate processing is analyzed in detail. Taking the chemical features of the FAHD1 substrate oxaloacetate into account, the potential inhibitor structures are deduced. The synthesis of drug-like scaffolds afforded first-generation FAHD1-inhibitors with activities in the low micromolar IC50 range. The investigations disclosed structures competing with the substrate for binding to the metal cofactor, as well as scaffolds, which may have a novel binding mode to FAHD1.

Deletion of the lactoperoxidase gene causes multisystem inflammation and tumors in mice

Strongly oxidative H2O2 is biologically important, but if uncontrolled, would lead to tissue injuries. Lactoperoxidase (LPO) catalyzes the redox reaction of reducing highly reactive H2O2 to H2O while oxidizing thiocyanate (SCN−) to relatively tissue-innocuous hypothiocyanite (OSCN−). SCN− is the only known natural, effective reducing-substrate of LPO; humans normally derive SCN− solely from food. While its enzymatic mechanism is understood, the actual biological role of the LPO-SCN− system in mammals remains unestablished. Our group previously showed that this system protected cultured human cells from H2O2-caused injuries, a basis for the hypothesis that general deficiency of such an antioxidative mechanism would lead to multisystem inflammation and tumors. To test this hypothesis, we globally deleted the Lpo gene in mice. The mutant mice exhibited inflammation and lesions in the cardiovascular, respiratory, digestive or excretory systems, neuropathology, and tumors, with high incidence. Thus, this understudied LPO-SCN− system is an essential protective mechanism in vivo.

Multiscale Simulations on the Catalytic Plasticity of CYP76AH1

The catalytic promiscuity and fidelity of cytochrome P450 enzymes are widespread in the skeletal modification of terpenoid natural products and have attracted much attention. CYP76AH1 is involved in key modification reactions in the biosynthetic pathway of tanshinone, a well-known medicinal norditerpenoid. In this work, classical molecular dynamic simulations, metadynamics, and DFT calculations were performed to investigate the protein conformational dynamics, ligand binding poses, and catalytic reaction mechanism in wide-type and mutant CYP76AH1. Our results not only reveal a plausible enzymatic mechanism for mutant CYP76AH1 leading to various products but also provide valuable guidance for rational protein engineering of the CYP76 family.

Possible Enzymatic Mechanism Underlying Chemical Tolerance and Characteristics of Tolerant Population in Scapholeberis Kingi

To determine the potential effects of pesticides on aquatic organisms inhabiting a realistic environment, we explored the characteristics and mechanisms of chemical tolerance in Scapholeberis kingi. We established a chemical-tolerant population via continuous exposure to pirimicarb, an acetylcholinesterase (AChE) inhibitor, and examined the effects of pirimicarb concentration on the intrinsic growth rates (r) of tolerant cladocerans. We also explored the association between r and feeding rate and tested the involvement of antioxidant enzymes [peroxidase (PO) and superoxide dismutase] and AChE in pirimicarb sensitivity. S. kingi was continuously exposed to sublethal pirimicarb concentrations (0, 2.5, 5, and 10 µg/L) for 15 generations and changes (half maximal effective concentration at 48 h, 48 h-EC50) in chemical sensitivity were investigated. In the F14 generation, the sensitivity of the 10 µg/L group was three times lower than that of the control group, suggesting the acquisition of chemical tolerance. Moreover, r was significantly and negatively correlated with 48 h-EC50, suggesting a fitness cost for tolerance. Surprisingly, there was no significant correlation between r and feeding rate. Our generalized linear model indicated that elevated PO activity may be related to chemical tolerance. Therefore, oxidative stress regulation may be involved in the acquisition of chemical tolerance in cladocerans. These findings will help elucidate the characteristics and mechanisms of chemical tolerance in aquatic organisms inhabiting a realistic environment.