Academic journal article Genetics

Onset of the DNA Replication Checkpoint in the Early Drosophila Embryo

Academic journal article Genetics

Onset of the DNA Replication Checkpoint in the Early Drosophila Embryo

Article excerpt

ABSTRACT

The Drosophila embryo is a promising model for isolating gene products that coordinate S phase and mitosis. We have reported before that increasing maternal Cyclin B dosage to up to six copies (six cycB) increases Cdk1-Cyclin B (CycB) levels and activity in the embryo, delays nuclear migration at cycle 10, and produces abnormal nuclei at cycle 14. Here we show that the level of CycB in the embryo inversely correlates with the ability to lengthen interphase as the embryo transits from preblastoderm to blastoderm stages and defines the onset of a checkpoint that regulates mitosis when DNA replication is blocked with aphidicolin. A screen for modifiers of the six cycB phenotypes identified 10 new suppressor deficiencies. In addition, heterozygote dRPA2 (a DNA replication gene) mutants suppressed only the abnormal nuclear phenotype at cycle 14. Reduction of dRPA2 also restored interphase duration and checkpoint efficacy to control levels. We propose that lowered dRPA2 levels activate Grp/Chk1 to counteract excess Cdk1-CycB activity and restore interphase duration and the ability to block mitosis in response to aphidicolin. Our results suggest an antagonistic interaction between DNA replication checkpoint activation and Cdk1-CycB activity during the transition from preblastoderm to blastoderm cycles.

EMBRYONIC development, except in mammals, is initiated by cleavage stages that are distinguished by rapid and "synchronous" cell cycles that are controlled by maternal gene products. Characteristically, these early cycles have only S and M phases (MURRAY and HUNT 1993). Both biochemical and morphological studies have documented that sequential activation and inactivation of Cdk1-CycB play a major role in regulating these biphasic cycles (EDGAR et al. 1994, 1995; SU et al. 1998; JI et al. 2004).

In Drosophila embryos, the first 13 mitoses, which occur without cytokinesis and thus produce a syncytium, are maternally controlled. Cell cycle length is not different during the first 6 of these cycles (9 min/cycle at 21°). In subsequent cycles, interphase length gradually extends (i.e., mitosis becomes delayed) from 200 sec in cycle 6, to 300 sec in cycle 10, to ~900 sec in cycle 13 (YU et al. 2000; JI et al. 2004). In addition, metaphase and anaphase, but not interphase durations, differ in different regions of the embryo, resulting in metasynchrony that correlates with local variation in Cyclin B (CycB) concentration (JI et al. 2004). Metasynchrony is also observed during the blastoderm cycles (cycles 10-13), when interphases become increasingly longer; however, at these stages the entire cell-cycle duration differs within the embryo. Nuclei at the poles divide faster (FOE and ALBERTS 1983; JI et al. 2004), which correlates with their lower nuclear densities (YASUDA 1992; BLANKENSHIP and WIESCHAUS 2001). Thus, metasynchronies at blastoderm are not a result of propagating mitotic waves.

We previously proposed that interphase extension in preblastoderm cycles (before cycle 10) occurs because CycB becomes limited. This conclusion is based on the following three observations: First, in later preblastoderm cycles (closer to cycle 10), when interphases become longer, embryos have less CycB than in cycle 6, presumably due to local degradation during each metaphase/anaphase transition (EDGAR et al. 1994; HUANG and RAFF 1999; STIFFLER et al. 1999). That is, CycB levels correlate inversely with interphase length. Second, increased CycB delays the onset of interphase extension; interphase extension occurs earlier in embryos from mothers with one copy of the cycB gene ("one cycB"; wild type has two copies) and later in embryos from mothers with four or six copies of cycB ("four cycB" or "six cycB" embryos; JI et al. 2002, 2004). Third, embryos from mothers lacking negative regulators of Cdk1-CycB, such as the DNA-replication checkpoint gene grapes (grp), which encodes a homolog of Chk1 kinase, exhibit interphase extensions not different from wild type during preblastoderm cycles (SIBON et al. …

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