RNA Primer Excision and Replacement by DNA polymerase I DNA polymerase III continues to synthesize DNA on the lagging strand until it is blocked by an RNA primer. DNA polymerase I excises the RNA primer in a 5’ to 3’ Exonuclease direction, and then fills the gap with a 5’ to 3’ Polymerase direction. It also proofreads the new chain using a 3’ to 5’ Exonuclease activity for DNA.3’-exonuclease is always for proofreading functions.DNA Ligase The Okazaki fragments are joined by DNA ligase, which forms the phosphodiester bond in the sugar phosphate backbone between the two nucleotides.The creation of the phosphodiester bond requires 2 high energy bonds.