Cdc6 activity analysis reveals possible unknown function in root meristem cells of Vicia faba

Abstract

Cell division cycle 6 (Cdc6) protein regulates the licensing of DNA replication, maintains centrosome stability, participates in DNA damage response (DDR) pathways and activates cell cycle checkpoints. The mechanisms responsible for Cdc6 relocation in the cell cycle are evolutionary conserved. Here, we analyzed changes in Cdc6 activity following hydroxyurea-induced replication stress and caffeine-induced premature chromosome condensation (PCC). We used Vicia faba root meristem cells as a model to explore the dynamics of activity changes of Cdc6, Chk1 and Chk2 proteins and their phosphorylated forms (Cdc6Ser54ph, Chk1Ser345ph and Chk2Thr68ph) under the conditions of prolonged replication stress. We observed the areas of increased Cdc6 activity that were not reported for animal cells: (i) the presence of clusters associated with low-density chromatin (euchromatin), (ii) the frequent vicinity of Cdc6 clusters to the most dense regions of constitutive heterochromatin. Cdc6 remains within the nucleus throughout the interphase which may suggest its new, yet unknown function in plants. The results of changes in Cdc6 activity during prolonged replicative stress also indicate that Cdc6 plays an important role in maintaining the cell cycle checkpoint in S phase when stress conditions last longer.