DNA end-joining catalyzed by human cell-free extracts

Mammalian cells defective in DNA end-joining are highly sensitive to ionizing radiation and are immunodeficient because of a failure to complete V(D)J recombination. By using cell-free extracts prepared from human lymphoblastoid cell lines, an in vitro system for end-joining has been developed. Intermolecular ligation was found to be accurate and to depend on DNA ligase IV/Xrcc4 and requires Ku70, Ku86, and DNA-PKcs, the three subunits of the DNA-activated protein kinase DNA-PK. Because these activities are involved in the cellular resistance to x-irradiation and V(D)J recombination, the development of this in vitro system provides an important advance in the study of the mechanism of DNA end-joining in human cells.

A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts

Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on in vitro-condensed chromosomes. The source of the kinetochore components is a clarified cell extract from Xenopus eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein–protein interactions involved in kinetochore assembly and function.

14-3-3 Proteins Act as Negative Regulators of the Mitotic Inducer Cdc25 in Xenopus Egg Extracts

Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2–cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3ε and to a lesser extent with 14-3-3ζ. The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G2–M transition.

Activation of the p42 Mitogen-activated Protein Kinase Pathway Inhibits Cdc2 Activation and Entry into M-Phase in Cycling Xenopus Egg Extracts

We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.