Loss of Cell Cycle Checkpoint Control in Drosophila Rfc4 Mutants

Abstract

Two alleles of the Drosophila melanogaster Rfc4 (DmRfc4) gene, which encodes subunit 4 of the replication factor C (RFC) complex, cause striking defects in mitotic chromosome cohesion and condensation. These mutations produce larval phenotypes consistent with a role in DNA replication but also result in mitotic chromosomal defects appearing either as premature chromosome condensation-like or precocious sister chromatid separation figures. Though the DmRFC4 protein localizes to all replicating nuclei, it is dispersed from chromatin in mitosis. Thus the mitotic defects appear not to be the result of a direct role for RFC4 in chromosome structure. We also show that the mitotic defects in these two DmRfc4 alleles are the result of aberrant checkpoint control in response to DNA replication inhibition or damage to chromosomes. Not all surveillance function is compromised in these mutants, as the kinetochore attachment checkpoint is operative. Intriguingly, metaphase delay is frequently observed with the more severe of the two alleles, indicating that subsequent chromosome segregation may be inhibited. This is the first demonstration that subunit 4 of RFC functions in checkpoint control in any organism, and our findings additionally emphasize the conserved nature of RFC's involvement in checkpoint control in multicellular eukaryotes.Control of cell cycle progression is critical for ensuring the precise duplication and distribution of the genome during cell division. One convenient way to envisage the cell cycle is as distinct phases, in which gap phases intervene before and between DNA replication and mitosis. However, a more accurate picture of the cell cycle emerges when the cell's ability to faithfully monitor crucial events is taken into account. Not only must the genome be completely and correctly replicated, but any remaining lesions in the sister chromatids must also be repaired prior to the entry into mitosis. Critically, the symmetric attachment of sister kinetochores to microtubules, achieving biorientation on the mitotic spindle, must occur prior to sister chromatid separation at the metaphase-to-anaphase transition. The concept of cell cycle checkpoints was initially proposed more than a decade ago with the characterization of the rad9 (for radiation sensitive) mutant in budding yeast (16). Since then, an explosion of studies of many diverse systems has served to illuminate three important checkpoints that normal cells progress through on the way to the completion of cell division: the DNA replication and DNA damage checkpoints (which may jointly be considered as DNA structure checkpoints) and the kinetochore attachment checkpoint. Precise molecular components have been identified in different model systems, primarily through genetic analysis of each of these checkpoints (6). Mutation of checkpoint components has been observed in many human cancers (9, 31).Though much of the original identification and analysis of cell cycle checkpoints was achieved with budding and fission yeast, studies with Drosophila me...

DNA Hypermethylation in Drosophila melanogaster Causes Irregular Chromosome Condensation and Dysregulation of Epigenetic Histone Modifications

“…The majority of structurally deviant chromosomes appeared overcondensed, and a smaller number revealed chromosome rearrangements and sister chromatid cohesion defects (data not shown). Intriguingly, the overall structural changes appeared similar to those seen with certain cell cycle mutants of D. melanogaster (33,36), which is consistent with our observation of delayed cell cycle progression. Lastly, a role of DNA methylation in the maintenance of structural chromosome integrity can also explain some of the mutations observed in mouse embryonic stem cells with a strongly demethylated genome (14).…”

The origin recognition complex is dispensable for endoreplication in Drosophila

“…2C), demonstrating a lack of DNA synthesis. These phenotypes represent proliferation defects, as was also observed in other replication initiation factor mutants, such as orc2, orc5, and Rfc4 (13)(14)(15). No imaginal discs were detectable in late third instar larvae, indicating that this tissue, which normally increases exponentially in cell number during larval development (16), has failed to proliferate.…”

The Spindle Pole Body Assembly Component Mps3p/Nep98p Functions in Sister Chromatid Cohesion

“…This latter model is supported by findings that mps3-1 mutant cells exhibit several phenotypes (including a mitotic delay) at the permissive temperature (3) and that elevated levels of Mps3p fail to rescue the essential function of Ctf7p (this study). Precedence for this model comes from studies involving Rfc5p, Rfc2p, Rfc4p, and Pol2p, which all perform essential roles in DNA replication but, when mutated, exhibit levels of cohesion loss similar to both mps3 mutant strains and nonessential cohesion factors (10,11,13,14). Our finding that a spindle pole body protein functions in cohesion is further supported by new evidence that Kar3p, a minus-end-directed spindle pole microtubule motor, participates in cohesion at levels similar to those found for Mps3p (20).…”