Resumen de Dinámica ORC-6 durante el ciclo celular en Arabidopsis thaliana
Initiation of genome replication in eukaryotes is a highly process that occurs at specific regions of the DNA (the origins of replication). The assembly of prereplicative complexes (pre-RC) at these origins is the first step for the initiation process. The origin recognition complex (ORC) iS a pre-RC component that acts as a landing pad for the rest of the pre-RC proteins. ORC6 is the smallest subunit of ORC. In Arabidopsis, ORC6 is not essential for plant viability as we demonstrate analyzing knock-down plant lines. We have studied the dynamics of Arabidopsis ORC6 during the cell cycle using GFP-tagged ORC6 genomic construct. We found that ORC6 is present in the nucleus during the S phase of the cell cycle but disappear late in G2 and during mitosis. We also show that ORC6 levels are highly regulated by the ubiquitin-proteasome system (UPS). We can rule out the possibility that CDK phosphorylation is implicated because Arabidopsis ORC6 does not contain any CDK consensus sites. Experiments using a dominant negative version of one subunit of casein kinase 2 (CK2) suggest that it might be involved in ORC6 phosphorylation. ORC6 is also subjected to proteasome-dependent degradation in response to other stimuli. One is in response to DNA damage, in particular to double strand breaks (DSB), but not to replication stress situations such as hydroxyurea. ORC6 degradation after DSB is associated with increased expression of G2 checkpoint marker genes and requires ATR, but not ATM. Another is in response to the hormone ethylene which induces a rapid degradation of ORC6 that occurs upstream of EIN3. We suggest that ORC6 may link these two processes. This hypothesis is supported by the phenotype observed in orc6 mutant with a constitutive ethylene phenotype and a constitutive up-regulation of genes involved in the G2 checkpoint response to DSB. Furthermore, the arabidopsis ctr1 mutant with an ethylene constitutive phenotype also has increased levels of the G2 checkpoint genes. Reduced levels of ORC6 induce the switch to the endocycle at early stages of leaf development as well as increased cell division in the root meristem. As an ORC component that participates in DNA replication we have been also able to show that it can be detected at sites where other ORC components, such as ORC1, bind, e.g. the CDT1 promoter, strongly suggesting that an origin of DNA replication may exist in this genomic location.
