Interactions of Claspin with replication and checkpoint proteins

The DNA damage and replication checkpoint ensures genome stability by monitoring DNA replication and the integrity of the genetic material. In the presence of DNA damage or replication stress, the checkpoint is activated. The activation of checkpoint grants time for cells to repair the damage and to overcome replication stress. Activation of the checkpoint requires phosphorylation of several important targets, such as checkpoint effector transcription factor p53 and checkpoint transducer kinase Chk1. The mediator protein Claspin was shown to be important in ATR-dependent Chk1 phosphorylation. Here I describe the purification and characterization of checkpoint- and replication-related domains of human Claspin. I have identified a minimal functional domain of Claspin necessary and sufficient for activation of ATR dependent Chk1 phosphorylation in vitro. Claspin has also been shown to bind to DNA that mimics the structure of replication fork. I identified a minimal DNA binding domain in Claspin. It has also been suggested that Claspin interacts with replication fork proteins. To understand which replication fork proteins Claspin directly interacts with, I have purified the following replication fork factors: Rad17-RFC, polymerase epsilon, Timeless and Cdc45. I then mapped the regions of Claspin involved in each protein-protein interaction using in vitro immunoprecipitation assays. My results suggest that Claspin interacts with the aforementioned replication fork proteins and replication fork DNA. This work provides new insights into the replication and checkpoint functions of Claspin and reconstitution of a well-defined minimal in vitro checkpoint system

High-resolution transcriptomic and epigenetic profiling identifies novel regulators of COPD

Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering its environmental cause, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) across COPD stages. We show that the epigenetic landscape is changed early in COPD, with DNA methylation changes occurring predominantly in regulatory regions. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Data integration identified 110 candidate regulators of disease phenotypes that were linked to fibroblast repair processes using phenotypic screens. Our study provides high-resolution multi-omic maps of HLFs across COPD stages. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic lung diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients

Mn(III) acetate-mediated regioselective benzylation of various alpha,beta-unsaturated and beta-alkoxy-alpha,beta-unsaturated ketones

We describe herein the results of manganese(III) acetate mediated alpha'-benzylation of various a,p-unsaturated and beta-alkoxy-alpha,beta-unsaturated ketones in moderate yields

Chemoenzymatic route to various spirocyclic compounds based on enantiomerically enriched tertiary allylic, homoallylic, and homopropargylic alcohols

A ring closing metathesis (RCM) reaction of dienes and an intramolecular Pauson-Khand (PKR) reaction of enynes derived from tertiary allyl, homoallyl, and homopropargyl alcohol backbones to afford the corresponding spirocyclic dihydrofuran and dihydropyrans and spirocyclic cyclopentenone pyrans, respectively, are described. Cyclopent-2-ene anchored tertiary allyl, homoallyl, and homopropargyl alcohols la-c have been efficiently resolved via enzymatic resolution with high ee (up to 90%) with 44%, 40%, and 43% chemical yields, respectively. Moreover, the cyclohex-2-ene anchored tertiary allyl, homoallyl, and homopropargyl alcohols 3a-c have also been resolved in the same manner with high ee (up to 97%) and in 42%, 45%, and 49% chemical yields. Enantiomerically enriched dienes derived from tertiary homoallyl alcohols yield the corresponding enantiomerically enriched spirocyclic dihydropyran derivatives via RCM with 74% and 78% chemical yields and with 90% and 97% ee, respectively. Moreover, enantiomerically enriched enynes derived from tertiary homoallyl alcohols afford the corresponding enantiomerically enriched cyclopentenone pyrans with spirocyclic motifs via PKR with 80% and 81% chemical yields, respectively, and as single diastereomers

Mn(III)-based C-C bond formation: regioselective alpha '-allylation of various alpha,beta-unsaturated, alpha and beta-alkoxy alpha,beta-unsaturated ketones

The Manganese(III) -based regioselective alpha'-keto radical generation of unsaturated ketones is a versatile synthetic procedure with broad applicability. The generated alpha'-keto radical slowly creates a metal enolate in a solvent at reflux. The resultant metal enolate affords the corresponding alpha'-allylated alpha,beta-unsaturated ketones in good yields. This method is the first example of the metal mediated regioselective alpha'-allylation of alpha,beta-unsaturated ketones. The ketones that have alpha or beta-alkoxy groups also work efficiently

The first enzymatic resolution of quaternary alpha '-acetoxy alpha,beta-unsaturated cyclohexenones and cyclopentenones

The enantio selective resolution of various quaternary alpha'-acetoxy alpha,beta-unsaturated cyclohexenones and cyclopentenones was performed with the commercially available enzyme CCL in pH = 8.0 phosphate buffer. Various parameters that would affect the enantioselectivities were tested and the best enzymatic resolution conditions were found to afford the enantiomerically enriched quaternary acetoxylated substrates with high ee varying between 36% and 99%

Enzyme-catalyzed resolution of aromatic ring fused cyclic tertiary alcohols

An efficient chemoenzymatic route for the synthesis of optically active aromatic ring fused cyclic tertiary alcohols (S)-(-)-1-methyl-1,2,3,4-tetrahydronaphthalen-1-ol (-)-1b and (S)-(+)-1-methyl-2,3-dihydro-1H-inden-1-ol (+)-1a has been developed. Different lipases have been tested in the transesterification of these tertiary alcohols; CAL-A (Candida antarctica Lipase A) was found to be the best biocatalyst for 1b and CAL-A-CLEA (Lipase A, C. antarctica, cross-linked enzyme aggregate) for la, obtained with ee values of 20% and 45%, respectively, and the corresponding esters 2b and 2a with the ee values of 99% and 71%, respectively

Conformational control on remote stereochemistry in the intramolecular Pauson-Khand reactions of enynes tethered to homoallyl and homopropargyl alcohols

An intramolecular Pauson-Khand reaction of enynes derived from homoallyl and homopropargyl alcohols is described. 2-Furyl substituted homoallyl and homopropargyl alcohols are easily and efficiently resolved through enzymatic resolution in a high ee (93-99%) with a known stereochemistry. Each enantiomerically enriched enyne affords the conformationally most stable diastereomeric cyclopenta[c]pyran ring system

CCL- and PLE-catalyzed reverse enantiomeric separation of various (+/-)-2-thienylcarbinols

(+/-)-2-Thienylcarbinols and their O-acetyl derivatives were resolved in reverse separation by CCL and PLE catalysed hydrolysis to afford optically active 2-thienylcarbinols in 35%-99% e.e. that are valuable chiral building blocks, in particular in the synthesis of pheromones and some alkoloid type natural products. Absolute configurations were determined by the correlation of specific rotation values with the literature and by transforming into the corresponding secondary alcohols via reductive desulfurization by Ra-Ni