Condensin is the core activity responsible for chromosome condensation in mitosis. In the yeast S. cerevisiae, condensin binding is enriched at the regions where DNA replication terminates. Therefore, we investigated whether DNA replication completion determines the condensin-binding proficiency of chromatin. In order to fulfill putative mitotic requirements for condensin activity we analyzed chromosome condensation and condensin binding to unreplicated chromosomes in mitosis. For this purpose we used pGAL:CDC6 cdc15-ts cells that are known to enter mitosis without DNA replication if CDC6 transcription is repressed prior to S-phase. Both the condensation of nucleolar chromatin and proper condensin targeting to rDNA sites failed when unreplicated chromosomes were driven in mitosis. We propose that the DNA replication results in structural and/or biochemical changes to replicated chromatin, which are required for two-phase condensin binding and proper chromosome condensation.
KeywordsDNA Replication; Chromosome Condensation; Condensin; rDNA; Nucleolus
INTRODUCTIONMitotic chromosome condensation is driven by consorted activities of structural chromatin proteins and enzymes, which enable a highly ordered compaction of sister chromatids into condensed chromosomes, thus preparing them for segregation. The condensin complex (1) is the major enzymatic activity required for this process (2,3). Condensin complex is present in all eukaryotic cells and is composed of five subunits: the Smc2/Smc4 heterodimer and three non-SMC subunits (1,4,5). In higher eukaryotes, there are two distinct sets of these non-SMC subunits, which form two condensin complexes (condensin I and II) upon association with the same SMC dimer (6,7). In vertebrates, the condensin-depleted sister chromatids are universally defective in separation during anaphase (8-10), as was originally reported in yeast (5,11).The molecular mechanisms of condensin activity remain obscure, however, mainly due to the inaccessibility of highly condensed chromatin in higher cells to biochemical and molecular analyses. Therefore, it is still unknown how condensin interacts with chromatin fiber -its Send correspondence to: Alexander V. Strunnikov, NIH, NICHD, LGRD, 18T Library Drive, Room 106, Bethesda, MD 20892, Tel: 301-402-8384, Fax: 301-402-1323, E-mail: E-mail: strunnik@mail.nih (12) suggests that condensin does not need to be regulated (e.g. by posttranslational modification) in order to display its basic enzymatic activity. Therefore, the regulation of condensin activity in vivo largely modulates condensin accessibility and affinity to chromatin, e.g. through nuclear envelope breakdown (6), nuclear import (4) or inhibitory phosphorylation (12). Directing condensin binding to specific (and probably invariant) chromatin sites (13,14) has to be an important component of this pathway, as judged from the highly ordered mitotic chromosome structure (and the corresponding regularity of the condensin binding pattern), which appears to be identical in every cell cycle...