DNA replication is the process whereby an entire double-stranded DNA is copied to produce a second, identical DNA double helix.
The objectives of this exercise are:
- To understand the functions of the proteins responsible for DNA replication, including helicase, SSB protein, primase, the sliding clamp, DNA polymerase, Rnase H and DNA ligase.
- to understand why the leading strand is synthesized continuously and the lagging strand is synthesized discontinuously.
DNA replication is an intricate process requiring the concerted action of many different proteins. The replication proteins are clustered together in particular locations in the cell and may therefore be regarded as a small “Replication Factory” that manufactures DNA copies. The DNA to be copied is fed through the factory, much as a reel of film is fed through a movie projector. The incoming DNA double helix is split into two single strands and each original single strand becomes half of a new DNA double helix. Because each resulting DNA double helix retains one strand of the original DNA, DNA replication is said to be semi-conservative.
DNA REPLICATION PROTEINS
DNA replication requires a variety of proteins. Each protein performs a specific function in the production of the new DNA strands.
- Helicase, made of six proteins arranged in a ring shape, unwinds the DNA double helix into two individual strands.
- Single-strand binding proteins, or SSBs, are tetramers that coat the single-stranded DNA. This prevents the DNA strands from reannealing to form double-stranded DNA.
- Primase is an RNA polymerase that synthesizes the short RNA primers needed to start the strand replication process.
- DNA polymerase is a hand-shaped enzyme that strings nucleotides together to form a DNA strand.
- The sliding clamp is an accessory protein that helps hold the DNA polymerase onto the DNA strand during replication.
- RNAse H removes the RNA primers that previously began the DNA strand synthesis.
- DNA ligase links short stretches of DNA together to create one long continuous DNA strand.
Let’s look at the steps of DNA replication in more detail. To begin the process of DNA replication, the two double helix strands are unwound and separated from each other by the helicase enzyme. The point where the DNA is separated into single strands, and where new DNA will be synthesized, is known as the replication fork. Single-strand binding proteins, or SSBs, quickly coat the newly exposed single strands. SSBs maintain the separated strands during DNA replication. Without the SSBs, the complementary DNA strands could easily snap back together. SSBs bind loosely to the DNA, and are displaced when the polymerase enzymes begin synthesizing the new DNA strands.
NEW STRAND SYNTHESIS
Now that they are separated, the two single DNA strands can act as templates for the production of two new, complementary DNA strands. Remember that the double helix consists of two antiparallel DNA strands with complementary 5’ to 3’ strands running in opposite directions. Polymerase enzymes can synthesize nucleic acid strands only in the 5’ to 3’ direction, hooking the 5’ phosphate group of an incoming nucleotide onto the 3’ hydroxyl group at the end of the growing nucleic acid chain. Because the chain grows by extension off the 3’ hydroxyl group, strand synthesis is said to proceed in a 5’ to 3’ direction.
Even when the strands are separated, however, DNA polymerase cannot simply begin copying the DNA. DNA polymerase can only extend a nucleic acid chain but cannot start one from scratch. To give the DNA polymerase a place to start, an RNA polymerase called primase first copies a short stretch of the DNA strand. This creates a complementary RNA segment, up to 60 nucleotides long that is called a primer.
Now DNA polymerase can copy the DNA strand. The DNA polymerase starts at the 3’ end of the RNA primer, and, using the original DNA strand as a guide, begins to synthesize a new complementary DNA strand. Two polymerase enzymes are required, one for each parental DNA strand. Due to the antiparallel nature of the DNA strands, however, the polymerase enzymes on the two strands start to move in opposite directions.
One polymerase can remain on its DNA template and copy the DNA in one continuous strand. However, the other polymerase can only copy a short stretch of DNA before it runs into the primer of the previously sequenced fragment. It is therefore forced to repeatedly release the DNA strand and slide further upstream to begin extension from another RNA primer. The sliding clamp helps hold this DNA polymerase onto the DNA as the DNA moves through the replication machinery. The sliding clamp makes the polymerase processive.
The continuously synthesized strand is known as the leading strand, while the strand that is synthesized in short pieces is known as the lagging strand. The short stretches of DNA that make up the lagging strand are known as Okazaki fragments.
THE LAGGING STRAND
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.