X-ray structure determination and analysis of DNA replication proteins derived from hyperthermophilic and psychrophilic archaea

The process of DNA replication in all organisms is complex and requires the concerted action of multiple proteins in order to achieve faithful replication of the genomic template. Since DNA replication is essential for the reproduction of an organism at a given temperature, proteins involved in this process likely have considerable selective pressure to adapt to their respective environments. We have determined the crystallographic structure of two classes of proteins central to the DNA replication machinery from a hyperthermophilic marine archaeon Thermococcus thioreducens and a psychrophilic archaeon Methanococcoides burtonii DSM 6242. The crystal structure of a family B DNA polymerase from T. thioreducens (TtPolB) has been solved to 2.0 A resolution, and the structure of the homotrimeric proliferating cell nuclear antigen (PCNA) from this organism (TtPCNA) has been solved to 1.9 A resolution. In addition, the structure of the PCNA from the psychrophilic archaeon M. burtonii DSM 6242 (MbPCNA) has been determined to 2.4 A resolution, and the DNA polymerase from this organism (MbPolB) has been cloned and overexpressed. The two PCNA structures are placed in the context of known sliding clamp structures from all domains of life, indicating high topological similarity despite low sequence identity. In contradiction to available literature, we demonstrate that the interfacial interactions responsible for maintaining a toroidal structure are not correlated with the temperature environment of the host organism, but rather are highly influenced by phylogeny. Our analysis indicates that the euryarchaeal PCNAs can be divided into two groups on the basis of interfacial interactions: one that relies on both charge-shape complementarity and one that is primarily stabilized by charge-charge complementarity. In addition, we demonstrate that the PCNA from the cold-adapted genome maintains its secondary structure well beyond the temperature associated with the optimal growth of the organism, indicating the presence of stable proteins in cold-adapted genomes. In contrast, the DNA polymerase from this organism demonstrates significant instability as it precipitates at temperatures extending into the mesophilic range. Structure-based sequence comparisons with TtPolB are performed to assess differences that may be related to their relative stabilities.