Relative stereochemical determination of the C61–C83 fragment of symbiodinolide using a stereodivergent synthetic approach

Structural determination is required in the use of marine natural products to create novel drugs and drug leads in medicinal chemistry. Symbiodinolide, which is a polyol marine natural product with a molecular weight of 2860, increases the intracellular Ca2+ concentration and exhibits inhibitory activity against cyclooxygenase-1. Seventy percent of the structure of symbiodinolide has been stereochemically clarified. Herein, we report the elucidation of the relative configuration of the C61–C83 fragment, which is among the remaining thirty percent, using a stereodivergent synthetic strategy. We first assigned the relative configuration of the C61–C74 fragment. Two candidate diastereomers of the C61–C74 fragment were synthesized, and their NMR data were compared with those of the natural product, revealing the relative stereochemistry of this component. We then narrowed down the candidate compounds for the C69–C83 fragment from 16 possible diastereomers by analyzing the NMR data of the natural product, and we thus selected eight candidate diastereomers. Stereodivergent synthesis of the candidates for this fragment and comparison of the NMR data of the natural product and the eight synthetic products resulted in the relative stereostructural clarification of the C69–C83 fragment. These individually determined relative stereochemistries of the C61–C74 and C69–C83 fragments were connected via the common C69–C73 tetrahydropyran moiety of the fragments. Finally, the relative configuration of the C61–C83 fragment of symbiodinolide was determined. The stereodivergent synthetic approach used in this study can be extended to the stereochemical determination of other fragments of symbiodinolide

p53 shapes genome-wide and cell type-specific changes in microRNA expression during the human DNA damage response.

The human DNA damage response (DDR) triggers profound changes in gene expression, whose nature and regulation remain uncertain. Although certain micro-(mi)RNA species including miR34, miR-18, miR-16 and miR-143 have been implicated in the DDR, there is as yet no comprehensive description of genome-wide changes in the expression of miRNAs triggered by DNA breakage in human cells. We have used next-generation sequencing (NGS), combined with rigorous integrative computational analyses, to describe genome-wide changes in the expression of miRNAs during the human DDR. The changes affect 150 of 1523 miRNAs known in miRBase v18 from 4-24 h after the induction of DNA breakage, in cell-type dependent patterns. The regulatory regions of the most-highly regulated miRNA species are enriched in conserved binding sites for p53. Indeed, genome-wide changes in miRNA expression during the DDR are markedly altered in TP53-/- cells compared to otherwise isogenic controls. The expression levels of certain damage-induced, p53-regulated miRNAs in cancer samples correlate with patient survival. Our work reveals genome-wide and cell type-specific alterations in miRNA expression during the human DDR, which are regulated by the tumor suppressor protein p53. These findings provide a genomic resource to identify new molecules and mechanisms involved in the DDR, and to examine their role in tumor suppression and the clinical outcome of cancer patients

A-type lamins maintain the positional stability of DNA damage repair foci in mammalian nuclei.

A-type lamins encoded by LMNA form a structural fibrillar meshwork within the mammalian nucleus. How this nuclear organization may influence the execution of biological processes involving DNA transactions remains unclear. Here, we characterize changes in the dynamics and biochemical interactions of lamin A/C after DNA damage. We find that DNA breakage reduces the mobility of nucleoplasmic GFP-lamin A throughout the nucleus as measured by dynamic fluorescence imaging and spectroscopy in living cells, suggestive of incorporation into stable macromolecular complexes, but does not induce the focal accumulation of GFP-lamin A at damage sites. Using a proximity ligation assay and biochemical analyses, we show that lamin A engages chromatin via histone H2AX and its phosphorylated form (γH2AX) induced by DNA damage, and that these interactions are enhanced after DNA damage. Finally, we use three-dimensional time-lapse imaging to show that LMNA inactivation significantly reduces the positional stability of DNA repair foci in living cells. This defect is partially rescued by the stable expression of GFP-lamin A. Thus collectively, our findings suggest that the dynamic structural meshwork formed by A-type lamins anchors sites of DNA repair in mammalian nuclei, providing fresh insight into the control of DNA transactions by nuclear structural organization

A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization.

Germline missense mutations affecting a single BRCA2 allele predispose humans to cancer. Here we identify a protein-targeting mechanism that is disrupted by the cancer-associated mutation, BRCA2(D2723H), and that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mislocalization of mutant BRCA2 inhibits the nuclear retention of RAD51 by exposing a similar NES in RAD51 that is usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Notably, BRCA2(D2723H) decreases RAD51 nuclear retention even when wild-type BRCA2 is also present. Our findings suggest a mechanism for the regulation of the nucleocytoplasmic distribution of BRCA2 and RAD51 and its impairment by a heterozygous disease-associated mutation

A-type lamins maintain the positional stability of DNA damage repair foci in mammalian nuclei.

A-type lamins encoded by LMNA form a structural fibrillar meshwork within the mammalian nucleus. How this nuclear organization may influence the execution of biological processes involving DNA transactions remains unclear. Here, we characterize changes in the dynamics and biochemical interactions of lamin A/C after DNA damage. We find that DNA breakage reduces the mobility of nucleoplasmic GFP-lamin A throughout the nucleus as measured by dynamic fluorescence imaging and spectroscopy in living cells, suggestive of incorporation into stable macromolecular complexes, but does not induce the focal accumulation of GFP-lamin A at damage sites. Using a proximity ligation assay and biochemical analyses, we show that lamin A engages chromatin via histone H2AX and its phosphorylated form (γH2AX) induced by DNA damage, and that these interactions are enhanced after DNA damage. Finally, we use three-dimensional time-lapse imaging to show that LMNA inactivation significantly reduces the positional stability of DNA repair foci in living cells. This defect is partially rescued by the stable expression of GFP-lamin A. Thus collectively, our findings suggest that the dynamic structural meshwork formed by A-type lamins anchors sites of DNA repair in mammalian nuclei, providing fresh insight into the control of DNA transactions by nuclear structural organization