Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork

Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM-Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly. We describe a method for inducing prolonged pausing of forks at protein barriers assembled at unique sites on a yeast chromosome, allowing us to examine for the first time the effects of pausing upon replisome integrity. We show that paused forks maintain an intact replisome that contains Mrc1, Tof1, MCM-Cdc45, GINS, and DNA polymerases alpha and epsilon and that recruits the Rrm3 helicase. Surprisingly, pausing does not require Mrc1, although Tof1 and Csm3 are both important. In addition, the integrity of the paused forks does not require Mec1, Rad53, or recombination. We also show that paused forks at analogous barriers in the rDNA are regulated similarly. These data indicate that paused and stalled eukaryotic replisomes resemble each other but are regulated differently.     

Abelson family kinases regulate Frizzled planar cell polarity signaling via Dsh phosphorylation

Abelson (Abl) family tyrosine kinases have been implicated in cell morphogenesis, adhesion, motility, and oncogenesis. Using a candidate approach for genes involved in planar cell polarity (PCP) signaling, we identified Drosophila Abl (dAbl) as a modulator of Frizzled(Fz)/PCP signaling. We demonstrate that dAbl positively regulates the Fz/Dishevelled (Dsh) PCP pathway without affecting canonical Wnt/Wg–Fz signaling. Genetic dissection suggests that Abl functions via Fz/Dsh signaling in photoreceptor R3 specification, a well-established Fz–PCP signaling readout. Molecular analysis shows that dAbl binds and phosphorylates Dsh on Tyr473 within the DEP domain. This phosphorylation event on Dsh is functionally critical, as the equivalent DshY473F mutant is nonfunctional in PCP signaling and stable membrane association, although it rescues canonical Wnt signaling. Strikingly, mouse embryonic fibroblasts (MEFs) deficient for Abl1 and Abl2/Arg genes also show reduced Dvl2 phosphorylation as compared with control MEFs, and this correlates with a change in subcellular localization of endogenous Dvl2. As in Drosophila, such Abl-deficient MEFs show no change in canonical Wnt signaling. Taken together, our results argue for a conserved role of Abl family members in the positive regulation of Dsh activity toward Fz–Dsh/PCP signaling by Dsh phosphorylation.