Step 1: Replication Fork Formation
Before DNA can be replicated, the double stranded molecule must be “unzipped” into two single strands. DNA has four bases called adenine [A], thymine [T], cytosine [C] and guanine [G] that form pairs between the two strands. Adenine only pairs with thymine and cytosine only binds with guanine. In order to unwind DNA, these interactions between base pairs must be broken. This is performed by an enzyme known as DNA helicase. DNA helicase disrupts the hydrogen bonding between base pairs to separate the strands into a Y shape known as the replication fork. This area will be the template for replication to begin.
DNA is directional in both strands, signified by a 5' and 3' end. This notation signifies which side group is attached the DNA backbone. The 5' end has a phosphate [P] group attached, while the 3' end has a hydroxyl [OH] group attached. This directionality is important for replication as it only progresses in the 5' to 3' direction. However, the replication fork is bi-directional; one strand is oriented in the 3' to 5' direction [leading strand] while the other is oriented 5' to 3' [lagging strand]. The two sides are therefore replicated with two different processes to accommodate the directional difference.
Step 2: Primer Binding
The leading strand is the simplest to replicate. Once the DNA strands have been separated, a short piece of RNA called a primer binds to the 3' end of the strand. The primer always binds as the starting point for replication. Primers are generated by the enzyme DNA primase.
Step 3: Elongation
Enzymes known as DNA polymerases are responsible creating the new strand by a process called elongation. There are five different known types of DNA polymerases in bacteria and human cells. In bacteria such as E. coli, polymerase III is the main replication enzyme, while polymerase I, II, IV and V are responsible for error checking and repair. DNA polymerase III binds to the strand at the site of the primer and begins adding new base pairs complementary to the strand during replication. In eukaryotic cells, polymerases alpha, delta, and epsilon are the primary polymerases involved in DNA replication. Because replication proceeds in the 5' to 3' direction on the leading strand, the newly formed strand is continuous. The lagging strand begins replication by binding with multiple primers. Each primer is only several bases apart. DNA polymerase then adds pieces of DNA, called Okazaki fragments, to the strand between primers. This process of replication is discontinuous as the newly created fragments are disjointed.
Step 4:
Termination
Once both the continuous and discontinuous strands are formed, an enzyme called exonuclease removes all RNA primers from the original strands. These primers are then replaced with appropriate bases. Another exonuclease “proofreads” the newly formed DNA to check, remove and replace any errors. Another enzyme called DNA ligase joins Okazaki fragments together forming a single unified strand. The ends of the linear DNA present a problem as DNA polymerase can only add nucleotides in the 5′ to 3′ direction. The ends of the parent strands consist of repeated DNA sequences called telomeres. Telomeres act as protective caps at the end of chromosomes to prevent nearby chromosomes from fusing. A special type of DNA polymerase enzyme called telomerase catalyzes the synthesis of telomere sequences at the ends of the DNA. Once completed, the parent strand and its complementary DNA strand coils into the familiar double helix shape. In the end, replication produces two DNA molecules, each with one strand from the parent molecule and one new strand.
| Before a cell divides, its DNA is replicated [duplicated.] Because the two strands of a DNA molecule have complementary base pairs, the nucleotide sequence of each strand automatically supplies the information needed to produce its partner. If the two strands of a DNA molecule are separated, each can be used as a pattern or template to produce a complementary strand. Each template and its new complement
together then form a new DNA double helix, identical to the original. Before replication can occur, the length of the DNA double helix about to be copied must beunwound. In addition, the two strands must beseparated, much like the two sides of a zipper, by breaking the weak hydrogen bonds that link the
paired bases. Once the DNA strands have been unwound, they must beheld apartto expose the bases so that new nucleotide partners can hydrogen-bond to them. |
Replication occurs differently on antiparallel strands of DNA.
The process starts with a short strand of DNA that binds by pairing its nucleotide bases to those in the DNA strand to be replicated. This "primer" has an exposed sugar molecule at its end. From there on, DNA polymerase can continuously synthesize the growing complementary strand. This strand of DNA is called the leading strand. A nice little animation of DNA synthesis on the leading strand can be seen at the Nobel Prize e-museum site at//www.nobel.se/medicine/educational/dna/a/replication/replication_ani.html.
On the complementary side of the DNA molecule, the primer would have a phosphate not a sugar at its exposed end; new nucleotides can only join to a sugar end. To get around this problem, this strand is synthesized in small pieces backward from the overall direction of replication. This strand is called the lagging strand. The short segments of newly assembled DNA from which the lagging strand is built are called Okazaki fragments. As replication proceeds and nucleotides are added to the sugar end of the Okazaki fragments, they come to meet each other. The whole thing is then stitched together by another enzyme called DNA ligase. The Nobel e-museum also has an animation of this process at //www.nobel.se/medicine/educational/dna/a/replication/lagging_ani.html .
Figure 2
Replication occurs simultaneously at multiple places along a DNA strand.
Because human DNA is so very long [with up to 80 million base pairs in a chromosome] it unzips at multiple places along its length so that the replication process is going on simultaneously at hundreds of places along the length of the chain. Eventually these areas run together to form a complete chain. In humans, DNA is copied at about50base pairs per second. The process would take a month [rather than the hour it actually does] without these multiple places on the chromosome where replication can begin.
DNA replication is extraordinarily accurate.
DNA polymerase makes very few errors, and most of those that are made are quickly corrected by DNA polymerase and other enzymes that "proofread" the nucleotides added into the new DNA strand. If a newly added nucleotide is not complementary to the one on the template strand, these enzymes remove the nucleotide and replace it with the correct one. With this system, a cell's DNA iscopied with less than one mistake in a billion nucleotides. This is equal to a person copying 100 large [1000 page] dictionaries word for word, and symbol for symbol, with only one error for the whole process!
Figure 1]
//www.genome.gov/Pages/Hyperion//DIR/VIP/Glossary/Illustration/dna_replication.shtml
Figure 2] //berget.mcs.cmu.edu/education/TechTeach/replication/purvCh21/RepFork.gif