mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos

Drosophila double park encodes a homolog of Cdt1 that functions in initiation of DNA replication in fission yeast and Xenopus [1-3], dup mutants complete the first 15 embryonic cell cycles, presumably via maternal dup products, and show defects in the 16(th) S phase (S16) [1]. Cells carrying dup(a1) allele forgo S16 altogether but enter mitosis 16 (M16) [1]. We find that the timing of entry into M16 is similar in dup(a1) and heterozygous or wildtype (wt) controls. In contrast, we find that mutant cells carrying another allele, dup(a3), undergo a partial S16 and delay the entry into M16. Thus, initiation of S16 appears necessary for delaying M16, This delay is absent in double mutants of dup(a3) and mei-41 (Drosophila ATR), indicating that a mei-41-dependent checkpoint acts to delay the entry into mitosis in response to incomplete DNA replication. dup(a3) and dup(a1) mutant cells that enter M16 become arrested in M16 [1]. We find that mitotic cyclins are stabilized and that a spindle checkpoint protein, Bub1, localizes onto chromosomes during mitotic arrest in dup mutants. These features suggest an arrest prior to metaphase-anaphase transition. dup(a3) bub1 double mutant cells exit M16, indicating that a bub1-mediated checkpoint acts to block mitotic exit in dup mutants. To our knowledge, this is the first report of (1) incomplete DNA replication affecting both the entry into and the exit from mitosis in a single cell cycle via different mechanisms and (2) the role of bub1 in regulating mitotic exit in response to incomplete DNA replication.