Glossary | Linus Pauling Institute | Oregon State University
The syntheses of histone, total protein, and DNA during the cell cycle were measured in the macronucleus of Euplotes eurystomus by assaying the incorporation of tritiated amino acids and tritiated thymidine in groups of 800 to 1000 synchronized cells. The synthesis of DNA begins at 30% completion of the cell cycle, proceeds at a constant rate, and ends very shortly before the beginning of macronuclear division. Histone labeling is absent during G1, begins in phase with DNA synthesis, continues at an unchanging rate during the S phase, and ends with the completion of DNA synthesis. The results support the view that the syntheses of histone and DNA are closely coupled events. Label in total protein accumulates at a constant rate during G1 and appears to shift to a slightly higher rate when histone synthesis begins. At division, radioactive DNA, histone, and total protein are distributed equally between the daughter macronuclei without loss of radioactivity. Radioautographic analysis showed that protein labeling occurs throughout the macronucleus during the entire life cycle. There was no clear difference in the degree of protein labeling between replicated and unreplicated regions of the macronucleus. The distribution of label suggests that most of macronuclear protein labeling during the cell cycle is concerned with the events of transcription rather than replication.
Amino Acid Synthesis and Metabolism
An aldol-based build/couple/pair (B/C/P) strategy was applied to generate a collection of stereochemically and skeletally diverse small molecules. In the phase, a series of asymmetric and aldol reactions were performed to produce four stereoisomers of a Boc-protected γ-amino acid. In addition, both stereoisomers of -PMB-protected alaninol were generated to provide a chiral amine coupling partner. In the step, eight stereoisomeric amides were synthesized by coupling the chiral acid and amine building blocks. The amides were subsequently reduced to generate the corresponding secondary amines. In the phase, three different reactions were employed to enable intramolecular ring-forming processes: nucleophilic aromatic substitution (SNAr), Huisgen [3+2] cycloaddition, and ring-closing metathesis (RCM). Despite some stereochemical dependencies, the ring-forming reactions were optimized to proceed with good to excellent yields, providing a variety of skeletons ranging in size from 8- to 14-membered rings. Scaffolds resulting from the RCM pairing reaction were diversified on the solid phase to yield a 14 400-membered library of macrolactams. Screening of this library led to the discovery of a novel class of histone deacetylase inhibitors, which display mixed enzyme inhibition, and led to increased levels of acetylation in a primary mouse neuron culture. The development of stereo-structure/activity relationships was made possible by screening all 16 stereoisomers of the macrolactams produced through the aldol-based B/C/P strategy.
In summary, we have shown that SIRT1-MT cells incorporate more BrdU than control cells under both normal and replicative stress conditions. Further analysis revealed that this is primarily due to the fact that the SIRT1-MT cells contain more active DNA replication origins and fail to arrest DNA synthesis even under replicative stress conditions. By analyzing SIRT1 interacting proteins as revealed by mass spectrometry analysis, we demonstrated that SIRT1 plays an essential role in replication fork firing and intra-S-phase checkpoint through deacetylating and regulating TopBP1. This study provides a molecular basis for the underlying mechanism that SIRT1 deficiency results in genetic instability.