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.

Control of division in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex

“…Immunoblotting with anti-GFP revealed a band at the expected size for the fusion following immunoprecipitation ( Fig 1A ) or direct Western blotting ( S1 Fig ). Levels of CYCB1-GFP were low in newborn cells and rose as cells began division cycles, similar to the pattern seen with CDKB1 and the replication control proteins CDC6, ORC1, MCM6 and CDC45 ([ 29 , 68 ]; S1 Fig ). This pattern of accumulation likely reflects strong transcriptional induction of these genes (along with essentially all other replication- or division-essential genes) in late G1 [ 43 , 44 ].…”

“…Increased accumulation of CYCB1-GFP in the absence of APC function ( Fig 1A ) suggested the likelihood that as in other systems, the APC promoted CYCB1 degradation. However, the imperfection of cell cycle synchrony in our hands is not good enough to resolve the individual rapid divisions, preventing determination of whether cyclin B was stable throughout the period of multiple fission and only degraded after all divisions were complete (as with MCM4 [ 68 ]), or was degraded in each division and then resynthesized.…”

“…To our knowledge, the problems we solved (here and in [ 68 ]) had inhibited or blocked previous description by time-lapse-fluorescence imaging of the complete Chlamydomonas multiple fission cycle.…”

Characteristics of N6-methyladenosine modification during sexual reproduction of Chlamydomonas reinhardtii

“…Many studies have shown that mitotic cell cycle has a long G1 phase and rapidly alternating S/M phases which facilitates Chlamydomonas to produce 2 n (n=1~5) daughter cells in one cell cycle [9,12]. In Chlamydomonas, the cell cycle and sexual reproduction is mainly controlled by two major cyclin dependent kinases of CDKA1 and CDKB1 [13,14], and other critical proteins reported previously [15,16]. The Chlamydomonas vegetative cells can be induced to produce two kinds of isogametes: mating type minus (mt+) and mating type plus (mt-) by nitrogen deficiency [10,[17][18][19], and many mating type specific genes are induced during this process [20][21][22].…”

The Papain-like Cysteine Protease HpXBCP3 from Haematococcus pluvialis Involved in the Regulation of Growth, Salt Stress Tolerance and Chlorophyll Synthesis in Microalgae

“…In plants and microalgae, proteases play essential roles in organism growth, reproduction, development, photosynthesis, senescence, and environmental stress responses [1,2]. For example, in Chlamydomonas reinhardtii, proteases are involved in the cell division [3]. In higher plants, papain-like cysteine proteases (PLCPs) are one of the major catalytic classes of protease [1].…”