SIRT6 protein deacetylase interacts with MYH DNA glycosylase, APE1 endonuclease, and Rad9-Rad1-Hus1 checkpoint clamp.

BACKGROUND: SIRT6, a member of the NAD(+)-dependent histone/protein deacetylase family, regulates genomic stability, metabolism, and lifespan. MYH glycosylase and APE1 are two base excision repair (BER) enzymes involved in mutation avoidance from oxidative DNA damage. Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp promotes cell cycle checkpoint signaling and DNA repair. BER is coordinated with the checkpoint machinery and requires chromatin remodeling for efficient repair. SIRT6 is involved in DNA double-strand break repair and has been implicated in BER. Here we investigate the direct physical and functional interactions between SIRT6 and BER enzymes. RESULTS: We show that SIRT6 interacts with and stimulates MYH glycosylase and APE1. In addition, SIRT6 interacts with the 9-1-1 checkpoint clamp. These interactions are enhanced following oxidative stress. The interdomain connector of MYH is important for interactions with SIRT6, APE1, and 9-1-1. Mutagenesis studies indicate that SIRT6, APE1, and Hus1 bind overlapping but different sequence motifs on MYH. However, there is no competition of APE1, Hus1, or SIRT6 binding to MYH. Rather, one MYH partner enhances the association of the other two partners to MYH. Moreover, APE1 and Hus1 act together to stabilize the MYH/SIRT6 complex. Within human cells, MYH and SIRT6 are efficiently recruited to confined oxidative DNA damage sites within transcriptionally active chromatin, but not within repressive chromatin. In addition, Myh foci induced by oxidative stress and Sirt6 depletion are frequently localized on mouse telomeres. CONCLUSIONS: Although SIRT6, APE1, and 9-1-1 bind to the interdomain connector of MYH, they do not compete for MYH association. Our findings indicate that SIRT6 forms a complex with MYH, APE1, and 9-1-1 to maintain genomic and telomeric integrity in mammalian cells.

The comprehensive antibiotic resistance database.

The field of antibiotic drug discovery and the monitoring of new antibiotic resistance elements have yet to fully exploit the power of the genome revolution. Despite the fact that the first genomes sequenced of free living organisms were those of bacteria, there have been few specialized bioinformatic tools developed to mine the growing amount of genomic data associated with pathogens. In particular, there are few tools to study the genetics and genomics of antibiotic resistance and how it impacts bacterial populations, ecology, and the clinic. We have initiated development of such tools in the form of the Comprehensive Antibiotic Research Database (CARD; http://arpcard.mcmaster.ca). The CARD integrates disparate molecular and sequence data, provides a unique organizing principle in the form of the Antibiotic Resistance Ontology (ARO), and can quickly identify putative antibiotic resistance genes in new unannotated genome sequences. This unique platform provides an informatic tool that bridges antibiotic resistance concerns in health care, agriculture, and the environment.

Multiomic spatial analysis reveals a distinct mucosa-associated virome.

The human gut virome has been increasingly explored in recent years. However, nearly all virome-sequencing efforts rely solely on fecal samples and few studies leverage multiomic approaches to investigate phage-host relationships. Here, we combine metagenomics, metaviromics, and metatranscriptomics to study virome-bacteriome interactions at the colonic mucosal-luminal interface in a cohort of three individuals with inflammatory bowel disease; non-IBD controls were not included in this study. We show that the mucosal viral population is distinct from the stool virome and houses abundant crAss-like phages that are undetectable by fecal sampling. Through viral protein prediction and metatranscriptomic analysis, we explore viral gene transcription, prophage activation, and the relationship between the presence of integrase and temperate phages in IBD subjects. We also show the impact of deep sequencing on virus recovery and offer guidelines for selecting optimal sequencing depths in future metaviromic studies. Systems biology approaches such as those presented in this report will enhance our understanding of the human virome and its interactions with our microbiome and our health.

Virome Sequencing of the Human Intestinal Mucosal-Luminal Interface.

While the human gut virome has been increasingly explored in recent years, nearly all studies have been limited to fecal sampling. The mucosal-luminal interface has been established as a viable sample type for profiling the microbial biogeography of the gastrointestinal tract. We have developed a protocol to extract nucleic acids from viruses at the mucosal-luminal interface of the proximal and distal colon. Colonic viromes from pediatric patients with Crohn's disease demonstrated high interpatient diversity and low but significant intrapatient variation between sites. Whole metagenomics was also performed to explore virome-bacteriome interactions and to compare the viral communities observed in virome and whole metagenomic sequencing. A site-specific study of the human gut virome is a necessary step to advance our understanding of virome-bacteriome-host interactions in human diseases.

Transformation of the Anticancer Drug Doxorubicin in the Human Gut Microbiome.

Bacteria living in the human gut are implicated in the etiology of several diseases. Moreover, dozens of drugs are metabolized by elements of the gut microbiome, which may have further implications for human health. Here, we screened a collection of gut isolates for their ability to inactivate the widely used antineoplastic drug doxorubicin and identified a strain of Raoultella planticola as a potent inactivator under anaerobic conditions. We demonstrate that R. planticola deglycosylates doxorubicin to metabolites 7-deoxydoxorubicinol and 7-deoxydoxorubicinolone via a reductive deglycosylation mechanism. We further show that doxorubicin is degraded anaerobically by Klebsiella pneumoniae and Escherichia coli BW25113 and present evidence that this phenotype is dependent on molybdopterin-dependent enzyme(s). Deglycosylation of doxorubicin by R. planticola under anaerobic conditions is found to reduce toxicity to the model species Caenorhabditis elegans, providing a model to begin understanding the role of doxorubicin metabolism by microbes in the human gut. Understanding the in vivo metabolism of important therapeutics like doxorubicin by the gut microbiome has the potential to guide clinical dosing to maximize therapeutic benefit while limiting undesirable side effects.