The objective of this research is to elucidate how eukaryotic DNA replication is coordinated and regulated under physiological DNA replication conditions. In eukaryotes, replication initiates at multiple sites of origin and on the lagging strand. Each initiation event must assemble a battery of proteins that will begin and complete with stringency, the process of DNA replication and elongation (reviewed in [1]). In eukaryotes this requires the systematized replacement of at least two DNA polymerases, and in the face of DNA lesion, further polymerase switching [1, 2].

The initiating polymerase, polymerase alpha (Pola), couples within its four-subunit complex both primase and DNA polymerase activities. The heterotetramer is conserved in all eukaryotes. The primase activity in Pola only known source of primase activity for DNA replication in eukaryotes. However, the polymerase activity of Pola lacks the ability to proofread and is non-processive. Pola only synthesizes a few nucleotides and must be replaced with a replicative polymerase, such as polymerases delta or epsilon (Pold or Pole) to both proofread and elongate(reviewed in [1]). How does this switch occur? The research in our lab is poised to answer these questions by examining these binding and exchange events on the DNA (See Figure 1). Specifically asking:

1. Will Pola bind and prime in the absence of the eukaryotic single-strand binding protein, RPA (replication promoting factor A), or as in vivo work suggests, require RPA, as well as RFC, the eukaryotic clamp loader (reviewed in [3, 4])?





2. Does proliferating cell nuclear antigen (PCNA), the DNA polymerase sliding clamp, displace Pola alone, or must PCNA load the replicative polymerase, Pold to displace Pola?
   a. Does PCNA loading of Pold occur on or off the DNA in the presence of Pola?
   b. Do multiple pathways exist where Pola can be displaced by either PCNA, or Pold, or a PCNA–Pold complex?
   

DNA replications are often not ideal [1, 2], and this research will segue well to probe situations where replication is stalled, and where DNA replicative (Pold) and repair (Polh) polymerases must exchange on damaged DNA to ensure the fidelity of replication (See Translesion Synthesis). This research will enhance the understanding of the order of events of DNA replication both under normal condition, and in the face of DNA damage and polymerase stalling and switching. The research will elucidate the triggers that are involved in replicative polymerase association, dissociation, repair polymerase binding, release, and replicative polymerase re-association, by examining these exchanges on template DNA. A detailed analysis of polymerase exchange will not only provide a better understanding of the ways people resist the deleterious effects of DNA damage, but may uncover possibilities for novel therapeutic treatments such as inhibition of the human translesion bypass (repair) polymerases. This may significantly enhance the effectiveness of chemotherapy treatments for cancer patients.