Equal distribution of genetic material during eukaryotic cell division requires reorganization of chromosome structure in mitosis, known as mitotic chromosome condensation. Condensation results in compaction of chromosomes, such that the average distance between points along the chromosome is reduced approximately fivefold in higher eukaryotes and twofold in budding yeast (reviewed by Koshland and Strunnikov, 1996
; Hirano, 1999
). Condensation is thought to serve several functions. These include the reduction of the length of chromosome arms such that they are shorter than half the length of the mitotic spindle and thus can be completely segregated into daughter cells during cytokinesis. Condensation may also help to resolve entangled chromatin fibers and increase mechanical resistance of the chromosomes to the forces of the mitotic spindle.
Several factors involved in this process have been identified in various organisms. They are evolutionarily related, as judged by their sequences, pointing to conservation of the basic mechanisms of mitotic chromosome condensation. These factors include a so-called condensin complex, topoisomerases, histone H3, and a number of additional proteins identified by yeast mutations.
The best biochemically characterized chromosome condensation factors are the 8S and 13S “condensin” complexes, identified in the Xenopus
egg extract system (Hirano et al., 1997
). The 8S complex is important, but not sufficient for mitotic chromosome condensation. It consists of two SMC-type (structural maintenance of chromosomes) proteins, XCAP-C and XCAP-E. Their budding yeast homologues, Smc2p and Smc4p, have also been implicated in chromosome condensation (Strunnikov et al., 1995
). The 13S condensin complex is necessary and sufficient to perform Xenopus
mitotic chromosome condensation in vitro. It consists of five subunits, which in addition to XCAP-C and XCAP-E include three unrelated proteins: XCAP-D2, XCAP-G, and XCAP-H (Hirano et al., 1997
). The 13S condensin complex is capable of binding DNA and using ATP to induce a global change in DNA configuration (Kimura and Hirano, 1997
; Kimura et al., 1999
In mitosis, XCAP-H, and to a lesser extent XCAP-G and XCAP-D2, subunits are hyperphosphorylated, and the complex is targeted to the chromosomes (Hirano et al., 1997
). Cdc2 protein kinase is at least partly responsible for this phosphorylation, which is accompanied by a shift in electrophoretic mobility of these proteins (Kimura et al., 1998
). This phosphorylation is necessary to activate the DNA reconfiguring activity of the condensin complex. It was hypothesized that this activity provides the driving force for mitotic DNA condensation (Hirano et al., 1997
; Kimura et al., 1999
). From the biochemical studies in Xenopus
, it appears that the function of condensins is limited to mitotic compaction of chromatin.
Mutations in fission yeast Schizosaccharomyces pombe
genes homologous to Xenopus
condensins cause defective chromosome condensation in mitosis (Sutani et al., 1999
). In this organism, mitotic phosphorylation of Cut3/SMC4 subunit, which is homologous to Xenopus
XCAP-C, is required for mitotic relocation of condensins from cytoplasm to the nucleus (Sutani et al., 1999
A mutation in the homologue of condensin subunit XCAP-H has been described in Drosophila
(Bhat et al., 1996
). It results in a mitotic chromosome segregation defect, in which the centromeres separate but chromosome arms do not get resolved. In contrast to the situation in Xenopus
egg extracts depleted of condensins, no detectable defect in chromosome condensation could be observed in the barren
mutant. The Barren protein was reported to interact with topoisomerase II and to activate its decatenating activity. It was hypothesized that the defect in topoisomerase II activation is responsible for the failure of chromosome resolution in mitosis in barren
mutant embryos (Bhat et al., 1996
Although chromosome condensation cannot be directly observed in budding yeast, it can be detected using fluorescence in situ hybridization (FISH), using either cosmid-size probes or probes that hybridize to the ribosomal DNA array (Guacci et al., 1994
). Ribosomal DNA encompasses a region of ~500 kb on chromosome XII, and its condensation state can be visually assessed after hybridization of a fluorescent probe. In interphase, the rDNA appears as a diffuse area, whereas in mitotic cells it has a defined string-like or bead-like shape (Guacci et al., 1994
A mutation in SMC2
, the budding yeast homologue of XCAP-E, leads to a defect in mitotic chromosome condensation and segregation (Strunnikov et al., 1995
). Mutant cells accumulate in mitosis while retaining relatively high viability. Some cells eventually undergo an abnormal division and arrest as unbudded cells (i.e., in the G1 phase of the cell cycle). When grown at permissive temperatures, the cells do not show a significant increase in the rate of chromosome loss. This set of characteristics is different in some respects from the phenotype of the yeast top2
mutants, which affect topoisomerase II (DiNardo et al., 1984
; Holm et al., 1989
). These cells attempt to segregate their chromosomes, which results in lethality. Unlike smc2
, the top2
mutant also has an increased chromosome loss rate.
Condensation defect was also detected in a double mutant trf4 top1
(Castano et al., 1996
was identified in a screen for mutations that are inviable in combination with topoisomerase I null mutation. Trf4p physically interacts with Smc1p and Smc2p. Its biochemical activities or cellular functions are unknown.
All five known Xenopus condensin subunits have highly similar homologues in the budding yeast genome. In addition to SMC2 and SMC4, there is BRN1, the homologue of the XCAP-H and Drosophila Barren, which is the focus of this work. We have also identified the yeast homologue of XCAP-G, YCG1, as a dosage suppressor of brn1 mutation. The homologue of XCAP-D2, named LOC7, was identified in a screen for genes necessary for sister chromatid separation and segregation (N. Bhalla and A. Murray [University of California, San Francisco, CA] Saccharomyces Genome Database entry). Here we explore the properties of BRN1 as a step to dissect the molecular mechanisms of mitotic chromosome condensation.