The chromosomes of eukaryotic cells contain cis-acting elements important for gene expression, replication, folding and structure, segregation and recombination. Among these regulatory sites only those involved in gene expression are well studied. The arrival of the Cas/CRISPR system for creating site directed mutations in chromosomes should dramatically increase the rate of discovery in these areas.
In eukaryotes each chromosome has many sites that serve as initiators for DNA replication in dividing cells. For multicellular eukaryotes the utilization of these sites changes during the course of development. The program of activation is poorly understood in metazoans and is epigenetic where proteins that control chromatin access rather than direct sequence recognition by the general replication factors is a dominant theme. Replication stress occurs when key replication factors are limiting as a result of hypomorphic mutation or when cells in a quiescent niche are induced to enter a division cycle through defaults in cell proliferation controls. Such stress may lead to chromosome breaks and accelerating damage drives cancer progression.
We purified the DmORC -complex (Drosophila origin recognition complex) first from extracts and were able to reconstitute the activity with recombinant proteins. This complex is part of the machinery that marks chromosomal DNA as sites "to be initiated" for replication. The execution point for ORC function is in G1 where a “latent helicase” activity is wrapped around the duplex. Conversion of the latent helicase into an active unwinding machine requires the association of 5 other proteins to this pre-replication assembly. In cells this conversion is the key switch step for S phase and regulated by the S phase promoting kinases. (Botchan Nature 2007). We have called this active form of the helicase the “CMG” (Ilves et al Mol Cell). How the latent helicase is converted to the active enzyme and how the CMG unwinds duplex is presently a key lab project. In other studies we are exploring cell cycle check-point controls that stop the unwinding under stress conditions. Recombinant ORC is now the focus of high-resolution structural studies to understand how ORC executes it’s key function with other proteins to load the latent helicase.
Beall et al (Nature 2002) first showed that a Myb protein-complex was a key factor in regulating site-specific DNA replication and this complex is now known to regulate transcription and DNA replication in different cell types. The core factors of this complex in turn recruit chromatin modifying factors to either repress or activate chromosome function. How the histone binding factor L(3)MBT is targeted to promoters and replication sites through the auspices of this complex is also a research direction.