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Human immunodeficiency virus (HIV) integrase inhibitors are increasingly being used for antiretroviral therapy (ART), and dolutegravir (DTG/Tivicay) has emerged as a leading core agent. In 2018, the Tsepamo study reported a 6-to 9-fold increase for neural tube defect (NTD) risk among the offspring of mothers receiving DTG during early gestation. Maternal folate (vitamin B9) status is the largest known modifier of NTD risk, so we evaluated folate-related mechanisms of action and the critical period for DTG developmental toxicity. Folate receptor (FOLR1) binding studies indicate DTG is a non-competitive FOLR1 antagonist at therapeutic concentrations. In vitro testing indicates calcium (2mM) increases FOLR1-folate interactions and alters DTG-FOLR1-folate interactions and cytotoxicity. DTG does not inhibit downstream folate metabolism by dihydrofolate reductase (DHFR). Early embryonic exposure toDTG is developmentally toxic in zebrafish, and supplemental folic acid can mitigate DTG developmental toxicity. The results from these studies are expected to inform and guide future animal models and clinical studies of DTG-based ART in women of childbearing age. The human immunodeficiency virus and acquired immune deficiency syndrome (HIV/AIDS) epidemic remains a prominent public health challenge. Globally, there are approximately 36.7 million people living with HIV, with 2.1 million new infections occurring annually, resulting in 1.1 million AIDS-related deaths per year 1 . Current HIV antiretroviral therapy (ART) includes C-C chemokine receptor type 5 (CCR5) antagonists, fusion inhibitors, integrase inhibitors, nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs), and protease inhibitors. Dolutegravir (DTG) is the clinically preferred integrase inhibitor used alone (Tivicay, ViiV Healthcare), or in combination (e.g. Triumeq, ViiV Healthcare) as ART for HIV infection in adults and children. A teratogenic risk for DTG was recently reported after neural tube defects (NTDs) were observed in four infants from mothers who had been taking DTG at the time of conception in Botswana 2 . The World Health Organization (WHO) subsequentlyprovided guidelines that DTG use be avoided by women of childbearing potential unless they used adequate contraception methods 3 . Based on this cohort study, the current guidelines are warranted and prudent, but more evidence is needed before scientific and medical communities can support a causal relationship between DTG and developmental toxicity, including NTDs. Developmental Toxicity of DTG:There is increasing usage of the HIV integrase inhibitors are for ART, and DTG has emerged as the leading compound in this class of medications. The DTG/Tivicay manufacturer reports (US FDA approved Tivicay Prescribing Information, 09/2018) animal reproduction studies showed no evidence of adverse developmental outcomes. Specifically, DTG was administered orally at up to 1,000 mg per kg daily, in rats and rabbits, during the period of organogenesis, days 6-17 and 6-18, respectively...

The ring-forming Hsp104 ATPase cooperates with Hsp70 and Hsp40 molecular chaperones to rescue stress-damaged proteins from both amorphous and amyloid-forming aggregates. The ability to do so relies upon pore loops present in the first ATP-binding domain (AAA-1; loop-1 and loop-2 ) and in the second ATP-binding domain (AAA-2; loop-3) of Hsp104, which face the protein translocating channel and couple ATP-driven changes in pore loop conformation to substrate translocation. A hallmark of loop-1 and loop-3 is an invariable and mutational sensitive aromatic amino acid (Tyr257 and Tyr662) involved in substrate binding. However, the role of conserved aliphatic residues (Lys256, Lys258, and Val663) flanking the pore loop tyrosines, and the function of loop-2 in protein disaggregation has not been investigated. Here we present the crystal structure of an N-terminal fragment of Saccharomyces cerevisiae Hsp104 exhibiting molecular interactions involving both AAA-1 pore loops, which resemble contacts with bound substrate. Corroborated by biochemical experiments and functional studies in yeast, we show that aliphatic residues flanking Tyr257 and Tyr662 are equally important for substrate interaction, and abolish Hsp104 function when mutated to glycine. Unexpectedly, we find that loop-2 is sensitive to aspartate substitutions that impair Hsp104 function and abolish protein disaggregation when loop-2 is replaced by four aspartate residues. Our observations suggest that Hsp104 pore loops have non-overlapping functions in protein disaggregation and together coordinate substrate binding, unfolding, and translocation through the Hsp104 hexamer.

The Ku heterodimer acts centrally in nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB). Ku, like mammalian Ku, binds and recruits NHEJ factors to DSB ends. Consequently, NHEJ is virtually absent in yeast Ku null (∆ or ∆) strains. Previously, we unexpectedly observed imprecise NHEJ proficiency in a yeast Ku mutant with impaired DNA end-binding (DEB). However, how DEB impairment supported imprecise NHEJ was unknown. Here, we found imprecise NHEJ proficiency to be a feature of a panel of DEB-impaired Ku mutants and that DEB impairment resulted in a deficiency in precise NHEJ. These results suggest that DEB-impaired Ku specifically promotes error-prone NHEJ. Epistasis analysis showed that classical NHEJ factors, as well as novel and previously characterized NHEJ-specific residues of Ku, are required for the distinct error-prone repair in a Ku DEB mutant. However, sequencing of repair junctions revealed that imprecise repair in Ku DEB mutants was almost exclusively characterized by small deletions, in contrast to the majority of insertions that define imprecise repair in wild-type strains. Notably, while sequencing indicated a lack of Pol4-dependent insertions at the site of repair, Pol2 exonuclease activity, which mediates small deletions in NHEJ, contributed to imprecise NHEJ in a Ku DEB mutant. The deletions were smaller than in Ku-independent microhomology-mediated end-joining (MMEJ) and were neither promoted by Mre11 nuclease activity nor Sae2 Thus, the quality of Ku's engagement at the DNA end influences end-processing during NHEJ and DEB impairment unmasks a Ku-dependent error-prone pathway of end-joining distinct from MMEJ.