5′→3′ Exonuclease activity: In addition to having the 5′→3′ polymerase activity that synthesizes DNA and the 3′→5′ exonuclease activity that proofreads the newly synthesized DNA like DNA pol III, monomeric DNA pol I also has a 5′→3′ exonuclease activity that is able to hydrolytically remove the RNA primer. [Note: Exonucleases remove nucleotides from the end of the DNA chain, rather than cleaving the chain internally as do endonucleases (Fig. 30.18).] First, DNA pol I locates the space (nick) between the 3′-end of the DNA newly synthesized by DNA pol III and the 5′-end of the adjacent RNA primer. Next, DNA pol I hydrolytically removes the RNA nucleotides ahead of itself, moving in the 5′→3′ direction (5′→3′ exonuclease activity). As it removes ribonucleotides, DNA pol I replaces them with deoxyribonucleotides, synthesizing DNA in the 5′→3′ direction (5′→3′ polymerase activity). As it synthesizes the DNA, it also proofreads using its 3′→5′ exonuclease activity to remove errors. This removal/synthesis/proofreading continues until the RNA primer is totally degraded, and the gap is filled with DNA (Fig. 30.19). [Note: DNA pol I uses its 5′→3′ polymerase activity to fill in gaps generated during most types of DNA repair (see p. 428).] 2.
Comparison of 5′→3′ and 3′→5′ exonuclease activities: The 5′→3′ exonuclease activity of DNA pol I allows the polymerase, moving 5′→3′, to hydrolytically remove one or more nucleotides at a time from the 5′-end of the ~10 nucleotide–long RNA primer. In contrast, the 3′→5′ exonuclease activity of DNA pol I and pol III allows these polymerases, moving 3′→5′, to hydrolytically remove one misplaced nucleotide at a time from the 3′-end of a growing DNA strand, increasing the fidelity of replication such that newly replicated DNA has one error per 107 nucleotides.
G. DNA ligase
The final phosphodiester linkage between the 5′-phosphate group on the DNA synthesized by DNA pol III and the 3′-hydroxyl group on the DNA made by DNA pol I is catalyzed by DNA ligase (Fig. 30.20). The joining of these two stretches of DNA requires energy, which in most organisms is provided by the cleavage of ATP to adenosine monophosphate + PPi.
H. Termination
Replication termination in E. coli is mediated by sequence-specific binding of the protein Tus (terminus utilization substance) to replication termination (ter) sites on the DNA, stopping the movement of the replication fork.
IV. EUKARYOTIC DNA REPLICATION
The process of eukaryotic DNA replication closely follows that of prokaryotic DNA synthesis. Some differences, such as the multiple origins of replication in eukaryotic cells versus single origins of replication in prokaryotes, have already been noted. Eukaryotic origin recognition proteins, ssDNA-binding proteins, and ATP-dependent DNA helicases have been identified, and their functions are analogous to those of the prokaryotic proteins previously discussed. In contrast, RNA primers are removed by RNase H and flap endonuclease 1 (FEN1) rather than by a DNA pol (Fig. 30.21).
A. Eukaryotic cell cycle