Control of pre-replicative complex during the division cycle in Chlamydomonas reinhardtii

Abstract

DNA replication is fundamental to all living organisms. In yeast and animals, it is triggered by an assembly of pre-replicative complex including ORC, CDC6 and MCMs. Cyclin Dependent Kinase (CDK) regulates both assembly and firing of the pre-replicative complex. We tested temperature-sensitive mutants blocking Chlamydomonas DNA replication. The mutants were partially or completely defective in DNA replication and did not produce mitotic spindles. After a long G1, wild type Chlamydomonas cells enter a division phase when it undergoes multiple rapid synchronous divisions (‘multiple fission’). Using tagged transgenic strains, we found that MCM4 and MCM6 were localized to the nucleus throughout the entire multiple fission division cycle, except for transient cytoplasmic localization during each mitosis. Chlamydomonas CDC6 was transiently localized in nucleus in early division cycles. CDC6 protein levels were very low, probably due to proteasomal degradation. CDC6 levels were severely reduced by inactivation of CDKA1 (CDK1 ortholog) but not the plant-specific CDKB1. Proteasome inhibition did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 might upregulate CDC6 at the transcriptional level. All of the DNA replication proteins tested were essentially undetectable until late G1. They accumulated specifically during multiple fission and then were degraded as cells completed their terminal divisions. We speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. We also developed a simple assay for salt-resistant chromatin binding of MCM4, and found that tight MCM4 loading was dependent on ORC1, CDC6 and MCM6, but not on RNR1 or CDKB1. These results provide a microbial framework for approaching replication control in the plant kingdom. The unicellular green alga Chlamydomonas reinhardtii undergoes cell division by ‘multiple fission’: repeated synchronous rounds of DNA replication and mitosis producing 8–16 daughter cells. In this study, we show that MCM proteins are localized to the nucleus throughout the multiple fission cycle, expect for transient loss during mitosis. CDC6 was also transiently localized to the nucleus. CDC6 levels were regulated by proteosomal protein degradation. We found evidence that the step-wise assembly of pre-replicative complex was conserved in Chlamydomonas. The plant-specific CDKB kinase was not required for this assembly. We propose a model that pre-RC assembly takes place right before the first replication cycle; assembly may be repeated in each cycle after the replication proteins are diffused in mitosis. This mechanism may contribute to timely replication in the multiple fission cycle.