THERMOCOCCUS KODAKARENSIS DNA REPLICATION MACHINERY

Abstract

DNA replication is the basis for the propagation and evolution of living organisms. It requires the combined efforts of numerous proteins. DNA replication in archaea has been shown to be more similar to eukarya than bacteria. Therefore, we use archaea as a model to study DNA replication. Euryarchaeon is one of the five main branches of archaeon. In this thesis, the replication machinery of the thermophilic euryarchaeon Thermococcus kodakarensis was investigated. In particular, this work focuses on two essential DNA replication proteins, the minichromosome maintenance (MCM) helicase and the processivity factor, proliferating cell nuclear antigen (PCNA). The MCM complex is thought to function as the replicative helicase in archaea and eukaryotes. In most archaea, one MCM homolog assembles to form the active homohexameric complex. Atypically, the genome of T. kodakarensis encodes three MCM homologs, here designated MCM1-3. Although all three MCM exhibit helicase activity, DNA binding and ATPase activities, only MCM3 appears to be essential for cell viability. Taken together with bioinformatics analysis, the results suggest that MCM3 is the replicative helicase in T. kodakarensis. PCNA is a ring shaped protein that encircles duplex DNA and, upon binding to the polymerase and other proteins, tethers them to the DNA. All euryarchaeal genomes, except T. kodakarensis, encode for a single PCNA protein. T. kodakarensis is unique because it contains two genes encoding for PCNA1 and PCNA2. It is shown here that both PCNA proteins stimulate DNA polymerase activity. It was found that PCNA1 is expressed in vivo at high levels in comparison to PCNA2. Furthermore, it was determined that PCNA2 is dispensable for cell viability. Taking together, the data presented herein suggest that T. kodakarensis is similar to other archaeal species studied, requiring only one MCM and one PCNA protein for viability. The results obtained from this work provide essential knowledge about the replication machinery in eukarya.