y, consisting of Pol? ,? , And? , Has been proposed. A protein is encoded in the plasmid pRN1 isolated from a Sulfolobus strain. This protein, ORF904 (named RepA), has primase and DNA polymerase activities in the N-terminal domain and helicase activity in the C-terminal domain, and is likely to be essential for the replication of pRN1. amino acid sequence of the N-terminal domain lacks homology to any known DNA polymerases or primases, and therefore, family E is proposed. Similar proteins are encoded by various archaeal and bacterial plasmids, as well as by some bacterial viruses. , One protein, tn2-12p, encoded in the plasmid pTN2 isolated from Thermococcus nautilus, was experimentally identified as a DNA polymerase in this family. This enzyme is likely responsible for the replication of the plasmids. Further investigations of this family of DNA polymerases will be interesting from an evolutional perspective.
10. PCNA and RFC
sliding clamp with the doughnut-shaped ring structure is conserved among living organisms, and functions as a platform or scaffold for proteins to work on the DNA strands. The eukaryotic and archaeal PCNAs form a homotrimeric ring structure, which encircles the DNA strand and anchors many important proteins involved in DNA replication and repair (Figure 4). works as a processivity factor that retains the DNA polymerase on the DNA by binding it on one surface (front side) of the ring for continuous DNA strand synthesis in DNA replication (Figure 5). To introduce the DNA strand into the central hole of the clamp ring, a clamp loader is required to interact with the clamp and open its ring. The archaeal and eukaryotic clamp loader is called RFC (Figure 5). most studied archaeal PCNA and RFC molecules to date are P. furiosus PCNA and RFC. The PCNA and RFC molecules are essential for DNA polymerase to perform processive DNA synthesis. The molecular mechanism of the clamp loading process has been actively investigated (Figure 5). intermediate PCNA-RFC-DNA complex, in which the PCNA ring is opened with out-of plane mode, was detected by a single particle analysis of electron microscopic images using P. furiosus proteins (Figure 6). crystal structure of the complex, including the ATP-bound clamp loader, the ring-opened clamp, and the template-primer DNA, using proteins from bacteriophage T4, has recently been published, and our knowledge about the clamp loading mechanism is continuously progressing.
Figure 4. PCNA-interacting proteins.
After clamp loading, DNA polymerase accesses the clamp and the polymerase-clamp complex performs processive DNA synthesis. Therefore, structural and functional analyses of the DNA polymerase-PCNA complex is the next target to elucidate the overall mechanisms of replication fork progression. The PCNA interacting proteins contain a small conserved sequence motif, called the PIP box, which binds to a common site on PCNA. The PIP box consists of the sequence Qxxhxxaa, where x represents any amino acid, h represents a hydrophobic residue (eg L, I or M), and a represents an aromatic residue (eg F, Y or W). Archaeal DNA polymerases have PIP box-like motifs in their sequences. However, only a few studies have experimentally investigated ...
