The availability of ARS elements, provided the opportunity for investigators to search for protein that interact with the ARS and to examine the steps involved in the initiation of eukaryotic DNA replication. This is a complex and rapidly developing field involving many different proteins. The discussion that follows is limited to the roles of four proteins that are required early in the initiation process, Many of the additional proteins that are required to complete the initiation stage help to regulate replication initiation and to ensure that he origins will fire only once during each cell cycle.
Stephen P. Bell and Bruce Stillman isolated the first protein, the origin recognition complex (ORC), in 1992 by taking advantage of the fact that ORC · ATP complexes bind to ARS elements. Specifically the ORC · ATP complex protects the A-element and part of the B1 element in ARST from DNase digestion. ORC consists of six different polypeptide subunits. Genes that code for these subunits have been cloned, facilitating genetic and biochemical studies.
Two lines of evidence suggest that ORC is a reasonable candidate for an eukaryotic initiator. First, yeast mutants with altered ORC subunits have a problem initiating chromosomal DNA synthesis and maintaining ARS plasmids. Second, nucleotide substitutions in the A-element, which reduce binding to ORC in vitro, also lower plasmid stability in vivo. ORC remains bound to yeast ARS throughout the yeast life cycle and helps to recruit three other proteins-Cdc6, Cdt1, and MCM to the ARSs as cells pass from the G1 to the S phase. The order of addition is Cdc6, Cdt1, and then MCM. The functions of these proteins are as follows：
Cdc6
Cdc6 is a cell division cycle (CDC) protein. The gene that codes for it, CDC6, was first identified by using a genetic screen designed to identify mutants that interfere with normal progression through the cell cycle. Yeast mutants that lack a functional Cdc6 do not replicate DNA. Biochemical studies reveal that Cdc6 helps to recruit Cdt1, Cdc6 is degraded by proteases once initiation is complete, helping to ensure that each origin first just one time during the cell cycle.
Cdt1
Yeast cells require Cdt1 to recruit MCM to the origin and to initiate the S phase.
MCM
MCM, a heterohexamer, is an ATP-dependent helicase that is essential for unwinding DNA during replication. Yeast mutants that produce nonfunctional MCM cannot initiate DNA synthesis. The gene that codes for MCM was first identified in mutants that could not maintain ARS plasmids. The MCM heterohexamer appears to work in the same way as the SV40 T antigen.
Additional proteins are recruited to the origin after MCM binds. These proteins include kinases that help to regulate replication initiation, the eukaryotic single-stranded DNA binding protein RPA, and three eukaryotic DNA polymerases.
