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 twoDmRfc4 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.

ACKNOWLEDGMENTS

We thank Maurizio Gatti for the l(3)e20 flies and for his encouragement in our endeavors to examine mutations affecting chromosome condensation. We are grateful to Bill Earnshaw and members of the Heck lab for lively discussions contributing to the understanding of chromosome structure. Neville Cobbe is thanked for his statistically significant skills. M.M.S.H. acknowledges Stuart MacNeill's insight into RFC phylogeny.

This work was supported by a Senior Research Fellowship in the Biomedical Sciences from the Wellcome Trust to M.M.S.H.

S. A. Krause and M.-L. Loupart contributed equally to this work.