RNA primer excision and replacement by DNA

DNA pol III continues to synthesize DNA on the lagging strand until it is blocked by proximity to an RNA primer. When this occurs, the RNA is excised and the gap filled by DNA pol I.
1. 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. 1).] 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. 1). [Note: DNA pol I uses its 5′→3′ polymerase activity to fill in gaps generated during most types of DNA repair.]

Figure 1:  Endonuclease versus exonuclease activity. [Note: Restriction endonucleases (see p. 481) cleave both strands.] T = thymine; A = adenine; C = cytosine; G = guanine.

Figure 2:  Removal of RNA primer and filling of the resulting gaps by DNA polymerase I.
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.