ists of two proteins, RFCS (small) and RFCL (large), in a 4 to 1 ratio. A different form of RFC, consisting of three subunits, RFCS1, RFCS2, and RFCL, in a 3 to 1 to 1 ratio, was also identified from M. acetivorans. The three subunits of RFC may represent an intermediate stage in the evolution of the more complex RFC in Eukaryota from the less complex RFC in Archaea.
Figure 6. Electron Microscopic Analysis of P. furious DNA polymerase-PCNA-DNA complex.
subunit organization and the spatial distribution of the subunits in the M. acetivorans RFC complex were analyzed and compared with those of the E. coli ?-Complex, which is also a pentamer consisting of three different proteins. These two clamp loaders adopt similar subunit organizations and spatial distributions, but the functions of the individual subunits are likely to be diverse.
11. DNA ligase
ligase is essential to connect the Okazaki fragments of the discontinuous strand synthesis during DNA replication, and therefore, it universally exists in all living organisms. This enzyme catalyzes phosphodiester bond formation via three nucleotidyl transfer steps. In the first step, DNA ligase forms a covalent enzyme-AMP intermediate, by reacting with ATP or NAD + as a cofactor. In the second step, DNA ligase recognizes the substrate DNA, and the AMP is subsequently transferred from the ligase to the 5-phosphate terminus of the DNA, to form a DNA-adenylate intermediate (AppDNA). In the final step, the 5-AppDNA is attacked by the adjacent 3-hydroxy group of the DNA and a phosphodiester bond is formed. DNA ligases are grouped into two families, according to their requirement for ATP or NAD + as a nucleotide cofactor in the first step reaction. ATPdependent DNA ligases are widely found in all three domains of life, whereas NAD +-dependent DNA ligases exist mostly in Bacteria. Some halophilic archaea and eukaryotic viruses also have NAD +-dependent enzymes. genes ( LIG1 , LIG3 and LIG4 ) encoding ATP-dependent DNA ligases have been identified in the human genome to date and DNA ligase I (Lig I), encoded by LIG1 , is a replicative enzyme that joins Okazaki fragments during DNA replication. The first gene encoding a eukaryotic-like ATP-dependent DNA ligase was found in the thermophilic archaeon, Desulfolobus ambivalens . Subsequent identifications of the DNA ligases from archaeal organisms revealed that these enzymes primarily use ATP as a cofactor. However, this classification may not be so strict. The utilization of NAD +, as well as ATP, as a cofactor has been observed in several DNA ligases, including those from T. kodakarensis , T. fumicolans, P. abyssi , Thermococcus sp. NA1, T. acidophilum, Picrophilus torridus, and Ferroplasma acidophilum, although ATP is evidently preferable in all of the cases. dual co-factor specificity (ATP / NAD +) is an interesting feature of these DNA ligase enzymes and it will be enlightening to investigate the structural basis for this...
