During DNA* replication a DNA double helix must unwind and separate so that DNA polymerase enzymes can use each single strand as a template for the synthesis of a new double strand. Strand separation is catalyzed by a Helicase* enzyme. A number of helper proteins prevent the strands from coming back together before replication is complete. Partial separation of the double helix forms a replication fork*.
The primary enzymes responsible for DNA replication are DNA polymerases1. Understanding the activity and limitations of DNA polymerases help in making sense of why DNA replication occurs the way it does. Key points are:
- Prokaryotic cells contain three different DNA polymerases. Each have slightly different activities. The two that are known to be required for DNA replication are DNA Polymerase III and DNA Polymerase I.
- DNA polymerases need a template. They can not synthesize a strand of DNA without it. As a result, DNA replication is semiconservative and depends on the presence of the two single strands of DNA formed at the replication fork.
- DNA polymerases can not create new polymers, they can only extend existing strands by adding new nucleotides* to one end. For replication to begin, another enzyme, Primase* ( a type of RNA polymerase), must create short priming sequences before DNA polymerases can begin their work. Unlike DNA polymerases, RNA polymerases can create new polymers by added based to a complimentary strand in the absence of an existing polymer. These short RNA polymer are called primers*. Once there is a short RNA primer, DNA polymerases can continue the elongation process.
- The RNA primers need to be removed prior to the end of the replication process. DNA polymerase I, which has exonuclease activity, performs this task.
- DNA polymerases can only add nucleotides to the 3'-hydroxyl end of a nucleotide polymer. Since the two strands of the original DNA helix are oriented in opposite directions. Double strand formation has to proceed in opposite directions on each of the two template strands at the replication fork.
In one direction DNA is replicated as one continuous strand. This is called the leading strand. Replication on the other strand occurs by the creation of many short segments. This is the lagging strand.
On both the leading and lagging strands, DNA replication starts with primase adding a short (3 to 10 base) RNA primer to the template strand. Once the primer is added, DNA polymerase III elongates the strand by added DNA nucleotides to the 3’-hydroxy end of the growing polymer.
These two steps are adequate to form long stretches of DNA on the leading strand.
On the lagging strand, the new strand's 3'-hydroxyl end points away from the replication fork. This forces the elongation process to occur in a discontinuous manner. As replication moves along the template strand, a series of shorter DNA polymers are formed. Each stretch is initiated with its own RNA primer. The shorter lengths of double stranded DNA formed along the lagging strand are called Okazaki fragments. When an Okazaki fragment extends to the point that it overlaps with the previous RNA primer, RNA nucleotides are removed and replaced by DNA. This requires DNA Polymerase I's exonuclease activity.
Once the RNA primer is completely by DNA the two DNA fragments are joined by a ligase enzyme.
1The process is quite complex and involves numerous enzymes and helper proteins. This discussion is limited to the major enzymes involved.↩
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