Synthetic Lethality between DNA Polymerase Epsilon and RTEL1 in Metazoan DNA Replication.

Genome stability requires coordination of DNA replication origin activation and replication fork progression. RTEL1 is a regulator of homologous recombination (HR) implicated in meiotic cross-over control and DNA repair in C. elegans.

Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis.

DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains.

Metabolism of DNA secondary structures at the eukaryotic replication fork.

DNA secondary structures are largely advantageous for numerous cellular processes but can pose specific threats to the progression of the replication machinery and therefore genome duplication and cell division. A number of specialized enzymes dismantle these structures to allow replication fork progression to proceed faithfully. In this review, we discuss the in vitro and in vivo data that has lead to the identification of these enzymes in eukaryotes, and the evidence that suggests that they act specifically at replication forks to resolve secondary structures.

GEN1/Yen1 and the SLX4 complex: Solutions to the problem of Holliday junction resolution.

Chromosomal double-strand breaks (DSBs) are considered to be among the most deleterious DNA lesions found in eukaryotic cells due to their propensity to promote genome instability. DSBs occur as a result of exogenous or endogenous DNA damage, and also occur during meiotic recombination. DSBs are often repaired through a process called homologous recombination (HR), which employs the sister chromatid in mitotic cells or the homologous chromosome in meiotic cells, as a template for repair.

Carbamate analogues of fumagillin as potent, targeted inhibitors of methionine aminopeptidase-2.

Inhibition of methionine aminopeptidase-2 (MetAP2) represents a novel approach to antiangiogenic therapy. We describe the synthesis and activity of fumagillin analogues that address the pharmacokinetic and safety liabilities of earlier candidates in this compound class. Two-step elaboration of fumagillol with amines yielded a diverse series of carbamates at C6 of the cyclohexane spiroepoxide.

Design, synthesis and selection of DNA-encoded small-molecule libraries.

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide.

Antiparasitic activities of novel, orally available fumagillin analogs.

Fumagillin, an irreversible inhibitor of MetAP2, has been shown to potently inhibit growth of malaria parasites in vitro. Here, we demonstrate activity of fumagillin analogs with an improved pharmacokinetic profile against malaria parasites, trypanosomes, and amoebas. A subset of the compounds showed efficacy in a murine malaria model.

Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.

Structure-specific endonucleases mediate cleavage of DNA structures formed during repair of collapsed replication forks and double-strand breaks (DSBs). Here, we identify BTBD12 as the human ortholog of the budding yeast DNA repair factor Slx4p and D. melanogaster MUS312.

Exogenous control of mammalian gene expression through modulation of RNA self-cleavage.

Recent studies on the control of specific metabolic pathways in bacteria have documented the existence of entirely RNA-based mechanisms for controlling gene expression. These mechanisms involve the modulation of translation, transcription termination or RNA self-cleavage through the direct interaction of specific intracellular metabolites and RNA sequences. Here we show that an analogous RNA-based gene regulation system can effectively be designed for mammalian cells via the incorporation of sequences encoding self-cleaving RNA motifs into the transcriptional unit of a gene or vector.

Exploration of the P6/P7 region of the peptide-binding site of the human class II major histocompatability complex protein HLA-DR1.

Crystal structures of the class II major histocompatibilty complex (MHC) protein, HLA-DR1, generally show a tight fit between MHC and bound peptide except in the P6/P7 region of the peptide-binding site. In this region, there is a shallow water-filled pocket underneath the peptide and between the pockets that accommodate the P6 and P7 side chains. We investigated the properties of this pocket with the idea of engineering substitutions into the corresponding region of peptide antigens to increase their binding affinity for HLA-DR1.