Before the lagging-strand DNA exits the replication factory, its RNA primers must be removed and the Okazaki fragments must be joined together to create a continuous DNA strand. The first step is the removal of the RNA primer. RNAse H, which recognizes RNA-DNA hybrid helices, degrades the RNA by hydrolyzing its phosphodiester bonds. Next, the sequence gap created by RNAse H is then filled in by DNA polymerase which extends the 3' end of the neighboring Okazaki fragment. Finally, the Okazaki fragments are joined together by DNA ligase that hooks together the 3' end of one fragment to the 5' phosphate group of the neighboring fragment in an ATP- or NAD+-dependent reaction.
REPLICATION IN ACTION
We are now ready to review the steps of DNA replication.
The process begins when the helicase enzyme unwinds the double helix to expose two single DNA strands and create two replication forks. DNA replication takes place simultaneously at each fork. The mechanism of replication is identical at each fork. Remember that the proteins involved in replication are clustered together and anchored in the cell. Thus, the replication proteins do not travel down the length of the DNA. Instead, the DNA helix is fed through a stationary replication factory like film is fed through a projector.
Single-strand binding proteins, or SSBs, coat the single DNA strands to prevent them from snapping back together. SSBs are easily displaced by DNA polymerase.
The primase enzyme uses the original DNA sequence as a template to synthesize a short RNA primer. Primers are necessary because DNA polymerase can only extend a nucleotide chain, not start one.
DNA polymerase begins to synthesize a new DNA strand by extending an RNA primer in the 5' to 3' direction. Each parental DNA strand is copied by one DNA polymerase. Remember, both template strands move through the replication factory in the same direction, and DNA polymerase can only synthesize DNA from the 5' end to the 3' end. Due to these two factors, one of the DNA strands must be made discontinuously in short pieces which are later joined together.
As replication proceeds, RNAse H recognizes RNA primers bound to the DNA template and removes the primers by hydrolyzing the RNA.
DNA polymerase can then fill in the gap left by RNase H.
The DNA replication process is completed when the ligase enzyme joins the short DNA pieces together into one continuous strand.