cDNA cloning and characterisation of a maize homologue of the MCM proteins required for the initiation of DNA replication

Abstract

A central question in cell cycle regulation is how DNA replication is initiated and executed only once in each cell cycle. The cell cycle-regulated assembly of specific initiation protein complexes at chromosomal origins appears to specify the initial sites and timing of DNA replication, and to restrict this process to only one round in the somatic cell cycle. Among the enzymes involved in origin activation, the MCM proteins play a conserved key role. In particular, MCM3 homologues have been shown to be components of the DNA replication licensing activity in yeast and vertebrates. In spite of our detailed knowledge of the regulation of the initiation of DNA synthesis in yeast, there is virtually no information available on the molecules involved in origin activation in higher plants. We have isolated a cDNA from maize root apices, termed ROA (Replication Origin Activator), encoding a protein which shares a high degree of homology with the MCM subfamily of MCM proteins. Analysis of gene organisation by Southern blotting shows 2-4 copies per haploid genome of closely related ROA sequences and the presence of further less related sequences in a multigene family. The steady-state levels of ROA mRNA are under developmental control, being relatively high in proliferative tissues such as the root apex, the developing cob and the coleoptile, and are strongly correlated with that of the histone H4 transcript. In situ hybridisation analysis in the root apex reveals that ROA mRNA expression is limited to specific subpopulations of cycling cells, which is typical of cell cycle-regulated expression. The isolation of nearly identical sequences from barley and Arabidopsis by the polymerase chain reaction indicates that MCM-related proteins are conserved in higher plants.