It is still to find out that how the actual replication initiation machinery is taken up by the replication origin. Here the actual replication initiation machinery can be replication protein-A or DNA polymerase a-primase.
The mechanism by which the components of the initiation complex such as Cdc45p or Mcm proteins become part of the elongating replication fork is still not clear.
It is still unclear that whether the protein-DNA interactions, found at the lamin B2 origin, are due to pre-RC (i.e. at the time of G1 phase) and post-RC (i.e. at the time of S phase) formation. But recent studies (RIP mapping analyses) show that the replication start points are close to the site of protein-DNA interaction.
It is still to be found whether Cdc6p and other proteins change origin recognition complex (ORC) binding in higher eukaryotes and whether this change affect the origin choice at the "Origin decision point" in mammalian cells.
It is still not clear whether ORCs of highly developed organisms recognize structural features or specific sequences.
The mechanism by which the iron rule of initiating DNA replication i.e. once and only once per cell cycle, predominates is still not known.
The relationship between replication start sites and the ORC binding sites is still unclear as the initiation pattern of the chorion origin at the nucleotide level is still to be determined. (Chorion is the outermost membrane around the embryo).
It is still to be found whether HoxC10p and HoxC13p (Proteins involved in morphogenesis of multicellular organisms) can bind to the ORC binding site.
Lagging strand (Okazaki fragment) start sites do not share any consensus sequence, even within a given gene position, and the mechanism underlying their regular spacing is still unclear.
Bielinsky, A. K., & Gerbi, S. A. (2001). Where it all starts: eukaryotic origins of DNA replication. Journal of Cell Science , 643-651.
Biology, Year 2001