The MCM-associated protein MCM-BP is important for human nuclear morphology

Abstract

SummaryMini-chromosome maintenance complex-binding protein (MCM-BP) was discovered as a protein that is strongly associated with human MCM proteins, known to be crucial for DNA replication in providing DNA helicase activity. The Xenopus MCM-BP homologue appears to play a role in unloading MCM complexes from chromatin after DNA synthesis; however, the importance of MCM-BP and its functional contribution to human cells has been unclear. Here we show that depletion of MCM-BP by sustained expression of short hairpin RNA (shRNA) results in highly abnormal nuclear morphology and centrosome amplification. The abnormal nuclear morphology was not seen with depletion of other MCM proteins and was rescued with shRNA-resistant MCM-BP. MCM-BP depletion was also found to result in transient activation of the G2 checkpoint, slowed progression through G2 and increased replication protein A foci, indicative of replication stress. In addition, MCM-BP depletion led to increased cellular levels of MCM proteins throughout the cell cycle including soluble MCM pools. The results suggest that MCM-BP makes multiple contributions to human cells that are not limited to unloading of the MCM complex.

A Role for USP7 in DNA Replication

“…In addition, a role for MCM-BP in unloading MCM complexes from chromatin at the end of S phase was identified using the Xenopus egg extract system, where MCM-BP depletion resulted in increased levels of MCM proteins on chromatin in mid-to late S phase and MCM-BP was shown to disrupt MCM complexes (23). Similar observations were subsequently made for MCM-BP in human cells (16,22,23). However, the mechanism by which MCM-BP regulates MCM complex unloading is unclear.…”

“…In human cells, we have shown that MCM-BP depletion results in altered nuclear morphology and replication stress, resulting in G 2 checkpoint activation (16). Similarly, inactivation or loss of the MCM-BP homologues in Schizosaccharomyces pombe (Mcb1) and Arabidopsis thaliana (ETG1) resulted in defects in DNA replication and G 2 checkpoint activation (17)(18)(19)(20).…”

The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication

“…Overexpression of the Mcb1 protein or Mcb1 inactivation in temperaturesensitive mcb1 mutants induces DNA damage and G 2 checkpoint activation (29,30). In human cells, the depletion of MCM-BP also leads to centrosome amplification and abnormal nuclear morphology, which may be due to G 2 DNA damage checkpoint activation (31). The loss of A. thaliana ETG1 leads to reduced DNA replication, activation of the G 2 checkpoint, and reduced sister chromatid cohesion (28,32).…”

“…The depletion of human MCM-BP also leads to reduced sister chromatid cohesion (32). The Xenopus MCM-BP appears to play a role in unloading MCM complexes from chromatin after DNA synthesis (27); however, the depletion of human MCM-BP not only increases the levels of chromatin-associated MCM proteins at the end of S-phase but also leads to a similar increase in soluble levels of MCM proteins throughout S-phase (31), suggesting multiple functions of MCM-BP in DNA replication. The human MCM-BP and A. thaliana ETG1 are largely nuclear throughout the cell cycle (28,31).…”

“…The Xenopus MCM-BP appears to play a role in unloading MCM complexes from chromatin after DNA synthesis (27); however, the depletion of human MCM-BP not only increases the levels of chromatin-associated MCM proteins at the end of S-phase but also leads to a similar increase in soluble levels of MCM proteins throughout S-phase (31), suggesting multiple functions of MCM-BP in DNA replication. The human MCM-BP and A. thaliana ETG1 are largely nuclear throughout the cell cycle (28,31). The fission yeast Mcb1 is widely distributed in the cytoplasm and nucleoplasm and is bound to chromatin (29); however, Xenopus MCM-BP is imported into the nucleus just before the dissociation of Mcm2-7 from chromatin near the end of S-phase.…”