Cell Cycle

Cell Cycle Control of DNA Replication Initiation of replication involves replicators and the origin recognition complex, or ORC, a heteromeric protein that binds to replicators. ORC is bound to the replicators throughout the cell cycle. Early in G1 (just after M), ORC serves as a “landing pad” for proteins essential to replication control. Proteins binding to ORC establish a pre-replication complex (pre-RC), but the pre-RC can be formed only during a window of opportunity during G1. One of the principal proteins in assembly of the pre-RC in yeast is Cdc6p (the replication activator protein encoded by the yeast cdc6 gene). Once Cdc6p binds to ORC, replication licensing factors (RLFs) that “license” or permit DNA replication to occur then bind to the chromosomes. www.web.virginia.edu/Heidi/chapter30/chp30.htm

Fluorescence Activated Cell Sorting (FACS) http://www.bio.davidson.edu/COURSES/GENOMICS/method/FACS.html

FLAG-Tag Plasmid (Example) HA-Tag: (hemagglutinin) YPYDVPDYA

PCNA (proliferating cell nuclear antigen) The protein encoded by this gene is found in the nucleus and is a cofactor of DNA polymerase. The encoded protein acts as a homotrimer and helps increase the processivity during DNA replication. http://biochemie.web.med.uni-muenchen.de/biotutor_2004/replikat.htm

DpnI cleaves DNA only when it is methylated on both strands. And mammalian cells lack the ability to methyate Dpn I restriction sites.