The role of bivalent ions in the regulation of D-loop extension mediated by DMC1 during meiotic recombination.

During meiosis, programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination. DMC1, a conserved recombinase, plays a central role in this process. DMC1 promotes DNA strand exchange between homologous chromosomes, thus creating the physical linkage between them. Its function is regulated not only by several accessory proteins but also by bivalent ions. Here, we show that whereas calcium ions in the presence of ATP cause a conformational change within DMC1, stimulating its DNA binding and D-loop formation, they inhibit the extension of the invading strand within the D-loop. Based on structural studies, we have generated mutants of two highly conserved amino acids - E162 and D317 - in human DMC1, which are deficient in calcium regulation. In vivo studies of their yeast homologues further showed that they exhibit severe defects in meiosis, thus emphasizing the importance of calcium ions in the regulation of DMC1 function and meiotic recombination.

Phosphorylation of the RecQ Helicase Sgs1/BLM Controls Its DNA Unwinding Activity during Meiosis and Mitosis.

The Bloom's helicase ortholog, Sgs1, orchestrates the formation and disengagement of recombination intermediates to enable controlled crossing-over during meiotic and mitotic DNA repair. Whether its enzymatic activity is temporally regulated to implement formation of noncrossovers prior to the activation of crossover-nucleases is unknown. Here, we show that, akin to the Mus81-Mms4, Yen1, and MutLγ-Exo1 nucleases, Sgs1 helicase function is under cell-cycle control through the actions of CDK and Cdc5 kinases. Notably, however, whereas CDK and Cdc5 unleash nuclease function during M phase, they act in concert to stimulate Sgs1 activity during S phase/prophase I. Mechanistically, CDK-mediated phosphorylation enhances the velocity and processivity of Sgs1, which stimulates DNA unwinding in vitro and joint molecule processing in vivo. Subsequent hyper-phosphorylation by Cdc5 appears to reduce the activity of Sgs1, while activating Mus81-Mms4 and MutLγ-Exo1. These findings suggest a concerted mechanism driving orderly formation of noncrossover and crossover recombinants in meiotic and mitotic cells.

The CHORD protein CHP-1 regulates EGF receptor trafficking and signaling in C. elegans and in human cells.

The intracellular trafficking of growth factor receptors determines the activity of their downstream signaling pathways. Here, we show that the putative HSP-90 co-chaperone CHP-1 acts as a regulator of EGFR trafficking in C. elegans. Loss of chp-1 causes the retention of the EGFR in the ER and decreases MAPK signaling. CHP-1 is specifically required for EGFR trafficking, as the localization of other transmembrane receptors is unaltered in chp-1(lf) mutants, and the inhibition of hsp-90 or other co-chaperones does not affect EGFR localization. The role of the CHP-1 homolog CHORDC1 during EGFR trafficking is conserved in human cells. Analogous to C. elegans, the response of CHORDC1-deficient A431 cells to EGF stimulation is attenuated, the EGFR accumulates in the ER and ERK2 activity decreases. Although CHP-1 has been proposed to act as a co-chaperone for HSP90, our data indicate that CHP-1 plays an HSP90-independent function in controlling EGFR trafficking through the ER.