N1 activity was also shown. crystal structure of the human FEN1-PCNA complex revealed three FEN1 molecules bound to each PCNA subunit of the trimer ring in different configurations. Based on these structural analyses together with the description in the DNA ligase section, a flip-flop transition mechanism, which enables proteins to internally switch for different functions on the same DNA clamp are currently being considered. The eukaryotic homologs of FEN1 were found in Archaea. crystal structures of FEN1 from M. jannaschii, P. furiosus , P. horikoshii , A. fulgidus , and S. solfataricus have been determined. In addition, detailed biochemical studies were performed on P. horikoshii FEN1. Thus, studies of the archaeal FEN1 proteins have provided important insights into the structural basis of the cleavage reaction of the flapped DNA. Our recent research showed that the flap endonuclease activity of P. furiosus FEN1 was stimulated by PCNA. , The stimulatory effect of PCNA on the sequential action of FEN1 and DNA ligase was observed in vitro (Kiyonari et al., Unpublished). Based on these results, a model of the molecular switching mechanisms of the last steps of Okazaki-fragment maturation was constructed. The quaternary complex of FEN1-Lig-PCNA-DNA was also isolated for the EM single particle analysis. These studies will provide more concrete image of the molecular mechanism.
13. Summary and perspectives
on the molecular mechanism of DNA replication has been a central theme of molecular biology. Archaeal organisms became popular in the total genome sequencing age, as described above, and most of the DNA replication proteins are now equally understood by biochemical characterizations. In addition, the archaeal studies are especially interesting to understand the mechanisms by which cells live in extreme environmental conditions. , It is also noteworthy that the proteins from the hyperthermophilic archaea are more stable than those from mesophilic organisms, and they are advantageous for the structural and functional analyses of higher-ordered complexes, such as the replisome. Studies on the higher-ordered complexes, rather than single proteins, are essential for understanding each of the events involved in DNA metabolism, and the archaeal research will continuously contribute to the development and advancement of the DNA replication research field, as summarized in part in a recent review. addition to basic molecular biology research, DNA replication proteins from thermophiles have been quite useful reagents for gene manipulations, including genetic diagnosis, forensic DNA typing, and detection of bacterial and virus infections, as well as basic research. Numerous enzymes have been commercialized around the world, and are utilized daily. An example of the successful engineering of an archaeal DNA polymerase for PCR is the creation of the fusion protein between P. furiosus Pol B and a nonspecific dsDNA binding protein, Sso7d, from S. solfataricus, by genetic engineering techniques. fusion DNA polymerase overcame the low processivity of the wild type Pol B by the high affinity Sso7d to the DNA strand. As another example, we s...
