In response to even a single chromosomal double-strand DNA break, cells enact the DNA damage checkpoint. This checkpoint triggers cell cycle arrest, providing time for the cell to repair damaged chromosomes before entering mitosis. This mechanism helps prevent the segregation of damaged or mutated chromosomes and thus promotes genomic stability. Recent work has elucidated the molecular mechanisms underlying several critical steps in checkpoint activation, notably the recruitment of the upstream checkpoint kinases of the ATM and ATR families to different damaged DNA structures and the molecular events through which these kinases activate their effectors. Chromatin modification has emerged as one important component of checkpoint activation and maintenance. Following DNA repair, the checkpoint pathway is inactivated in a process termed recovery. A related but genetically distinct process, adaptation, controls cell cycle re-entry in the face of unrepairable damage.

One of the earliest marks of a double-strand break (DSB) in eukaryotes is serine phosphorylation of the histone variant H2AX at the carboxy-terminal SQE motif to create gammaH2AX-containing nucleosomes. Budding-yeast histone H2A is phosphorylated in a similar manner by the checkpoint kinases Tel1 and Mec1 (ref. 2; orthologous to mammalian ATM and ATR, respectively) over a 50-kilobase region surrounding the DSB. This modification is important for recruiting numerous DSB-recognition and repair factors to the break site, including DNA damage checkpoint proteins, chromatin remodellers and cohesins. Multiple mechanisms for eliminating gammaH2AX as DNA repair completes are possible, including removal by histone exchange followed potentially by degradation, or, alternatively, dephosphorylation. Here we describe a three-protein complex (HTP-C, for histone H2A phosphatase complex) containing the phosphatase Pph3 that regulates the phosphorylation status of gammaH2AX in vivo and efficiently dephosphorylates gammaH2AX in vitro. gammaH2AX is lost from chromatin surrounding a DSB independently of the HTP-C, indicating that the phosphatase targets gammaH2AX after its displacement from DNA. The dephosphorylation of gammaH2AX by the HTP-C is necessary for efficient recovery from the DNA damage checkpoint.

Mesodermal progenitors arise in theDuring animal development, a wide diversity of cellular identities must be specified within initially undifferentiated fields of cells. One solution to this problem is for a hierarchy of regulators to promote the progressive determination of cells, essentially carving out from the original field domains with increasingly restricted developmental potential. In such a mechanism, spatially localized factors first delineate a prepattern in which all cells are equally competent to adopt a particular identity (Stern 1954;Greenwald and Rubin 1992). The expression of additional regulatory molecules in cellular subsets within the prepatterned territory further limits the responses afforded particular cells. Precise refinement of the final pattern can be dictated by direct inhibitory interactions among neighboring cells (Greenwald and Rubin 1992;Simpson 1997). Although many details are known about the later pattern forming steps in a number of developmental systems, relatively little information is available for how early prepatterns are established (Greenwald and Rubin 1992;Kornfeld 1997;Simpson 1997;Vervoort et al. 1997).The Drosophila embryonic mesoderm provides an ideal system in which to investigate prepattern and pattern formation. The mesoderm arises from the ventral most cells of the blastoderm embryo under the influence of the zygotic genes, twist (twi) and snail (sna). Cells expressing these genes invaginate through the ventral furrow at gastrulation. Subsequently, the internalized mesodermal cells migrate dorsolaterally to form a uniform sheet beneath the ectoderm (Bate 1993;Leptin 1995), a process that is controlled by a fibroblast growth factor (FGF) receptor encoded by heartless (htl;Beiman et al. 1996;Gisselbrecht et al. 1996;Shishido et al. 1997;Michelson et al. 1998).

In many cells the timing of entry into mitosis is controlled by the balance between the activity of inhibitory Wee1-related kinases (Swe1p in budding yeast) and the opposing effect of Cdc25-related phosphatases (Mih1p in budding yeast) that act on the cyclin-dependent kinase Cdc2 (Cdc28p in budding yeast). Wee1 and Cdc25 are key elements in the G2 arrest mediated by diverse checkpoint controls. In budding yeast, a 'morphogenesis checkpoint' that involves Swe1p and Mih1p delays mitotic activation of Cdc28p. Many environmental stresses (such as shifts in temperature or osmolarity) provoke transient depolarization of the actin cytoskeleton, during which bud construction is delayed while cells adapt to environmental conditions. During this delay, the morphogenesis checkpoint halts the cell cycle in G2 phase until actin can repolarize and complete bud construction, thus preventing the generation of binucleate cells. A similar G2 delay can be triggered by mutations or drugs that specifically impair actin organization, indicating that it is probably actin disorganization itself, rather than specific environmental stresses, that triggers the delay. The G2 delay involves stabilization of Swe1p in response to various actin perturbations, although this alone is insufficient to produce a long G2 delay.