DNA replication is the process of copying a double-stranded deoxyribonucleic acid (DNA) molecule, a process essential in all known life forms. The general mechanisms of DNA replication are different in prokaryotic and eukaryotic organisms.
As each DNA strand holds the same genetic information, both strands can serve as templates for the reproduction of the opposite strand. The template strand is preserved in its entirety and the new strand is assembled from nucleotides. This process is called semiconservative replication. The resulting double-stranded DNA molecules are identical; proofreading and error-checking mechanisms exist to ensure extremely high fidelity.
In a cell, DNA replication must happen before cell division. Prokaryotes replicate their DNA throughout the interval between cell divisions. In eukaryotes, timings are highly regulated and this occurs during the S phase of the cell cycle, preceding mitosis or meiosis I.
DNA structure
A DNA strand is a long polymer built from nucleotides; two complementary DNA strands form a double helix, each strand possessing a 5' phosphate end and a 3' hydroxyl end. The numbers followed by the prime indicate the position on the deoxyribose sugar backbone to which the phosphate or hydroxyl group is attached (numbers without primes are reserved for the bases). The two strands in the DNA backbone run anti-parallel to each other: One of the DNA strands is built in the 5' → 3' direction while the other runs in an anti parallel direction, although its information is stored in the 3' → 5' direction. Each nucleotide consists of a phosphate, a simple sugar or a deoxyribose sugar - forming the backbone of the DNA double helix - plus a base. The bonding angles of the backbone ensures that DNA will tend to twist as the length of the molecule progresses, giving rise a double helix shape instead of a straight ladder. Base pairs form the steps of the helix ladder while the sugars and phosphate molecules forms the handrail. Each of the four bases has a partner to which it makes the strongest hydrogen bonds. When a nucleotide base forms hydrogen bonds with its complementary base on the other strand, they form a base pair: Adenine pairs with thymine and cytosine pairs with guanine. These pairings can be expressed as C•G and A•T, or C≡G and A=T where the number of lines indicate the number of hydrogen bonds between each base pair. For example, a 10-base pair strand running in the 5' → 3' direction that has adenine as the 3rd base will pair with the base thymine as the 7th base on the complementary 10-base pair strand running in the opposite direction.
The replication fork
The replication fork is a structure which forms when DNA is being replicated. It is created through the action of helicase, which breaks the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching "prongs", each one made up of a single strand of DNA.
Lagging strand synthesis
In DNA replication, the lagging strand is the DNA strand at the replication fork opposite to the leading strand. It is also oriented in the opposite direction when compared to the leading strand, with the 5' near the replication fork instead of the 3' end as is the case with the leading strand. When the enzyme helicase unwinds DNA, two single stranded regions of DNA (the "replication fork") form. DNA polymerase cannot build a strand in the 3' → 5' direction. This poses no problems for the leading strand, which can continuously synthesize DNA in a processive manner, but creates a problem for the lagging strand, which cannot be synthesized in the 3' → 5' direction. Thus, the lagging strand is synthesized in short segments known as Okazaki fragments. On the lagging strand, primase builds an RNA primer in short bursts. DNA polymerase is then able to use the free 3' hydroxyl group on the RNA primer to synthesize DNA in the 5' → 3' direction. The RNA fragments are then removed (different mechanisms are used in eukaryotes and prokaryotes) and new deoxyribonucleotides are added to fill the gaps where the RNA was present. DNA ligase is then able to join the deoxyribonucleotides together, completing the synthesis of the lagging strand.
Leading strand synthesis
The leading strand is defined as the DNA strand that is read in the 3' → 5' direction but synthesized in the 5'→ 3' direction, in a continuous manner. On this strand, DNA polymerase III is able to synthesize DNA using the free 3'-OH group donated by a single RNA primer (multiple RNA primers are not used) and continuous synthesis occurs in the direction in which the replication fork is moving.
Dynamics at the replication fork
The sliding clamp in all domains of life share a similar structure, and are able to interact with the various processive and non-processive DNA polymerases found in cells. In addition, the sliding clamp serves as a processivity factor. The C-terminal end of the clamps forms loops which are able to interact with other proteins involved in DNA replication (such as DNA polymerase and the clamp loader). The inner face of the clamp allows DNA to be threaded through it. The sliding clamp forms no specific interactions with DNA. There is a large 35A hole in the middle of the clamp. This allows DNA to fit through it, and water to take up the rest of the space allowing the clamp to slide along the DNA. Once the polymerase reaches the end of the template or detects double stranded DNA (see below), the sliding clamp undergoes a conformational change which releases the DNA polymerase.
The clamp loader, a multisubunit protein, is able to bind to the sliding clamp and DNA polymerase. When ATP is hydrolyzed, it loses affinity for the sliding clamp allowing DNA polymerase to bind to it. Furthermore, the sliding clamp can only be bound to a polymerase as long as single stranded DNA is being synthesized. Once the single stranded DNA runs out, the polymerase is able to bind to the a subunit on the clamp loader and move to a new position on the lagging strand. On the leading strand, DNA polymerase III associates with the clamp loader and is bound to the sliding clamp.
Recent evidence suggests that the enzymes and proteins involved in DNA replication remain stationary at the replication forks while DNA is looped out to maintain bidirectionality observed in replication. This is a result of an interaction between DNA polymerase, the sliding clamp, and the clamp loader.
DNA replication
DNA replication differs somewhat between eukaryotic and prokaryotic cells. Much of our knowledge of prokaryotic DNA replication was derived from the study of E. coli, while yeast has been used as a model organism for understanding eukaryotic DNA replication.
Mechanism of replication
Once priming of DNA is complete, DNA polymerase is loaded into the DNA and replication begins. The catalytic mechanism of DNA polymerase involves the use of two metal ions in the active site and a region in the active site that can discriminate between deoxyribonucleotides and ribonucleotides. The metal ions are general divalent cations that help the 3'-OH initiate a nucleophilic attack on the alpha-phosphate of the deoxyribonucleotide and orient and stabilize the negatively charged triphosphate on the deoxyribonucleotide. Nucleophilic attack by the 3'-OH on the alpha phosphate releases pyrophosphate, which is then subsequently hydrolyzed by inorganic pyrophosphatase into two phosphates. This hydrolysis drives DNA synthesis to completion.
Furthermore, DNA polymerase must be able to distinguish between correctly paired bases and incorrectly paired bases. This is accomplished by distinguishing Watson-Crick base pairs through the use of an active site pocket that is complementary in shape to the structure of correctly paired nucleotides. This pocket has a tyrosine residue that is able to form van der Waals interactions with the correctly paired nucleotide. In addition, double stranded DNA in the active site has a wider and shallower minor groove that permits the formation of hydrogen bonds with the third nitrogen of purine bases and the second oxygen of pyrimidine bases. Finally, the active site makes extensive hydrogen bonds with the DNA backbone. These interactions result in the DNA polymerase III closing around a correctly paired base. If a base is inserted and incorrectly paired, these interactions could not occur due to disruptions in hydrogen bonding and van der Waals interactions. The mechanism of replication is similar in eukaryotes and prokaryotes.
DNA is read in the 3' → 5' direction, relative to the parent strand, therefore, nucleotides are synthesized (or attached to the template strand) in the 5' → 3' direction, relative to the daughter strand. However, one of the parent strands of DNA is 3' → 5' and the other is 5' → 3'. To solve this, replication must proceed in opposite directions. The leading strand runs towards the replication fork and is thus synthesized in a continuous fashion, only requiring one primer. On the other hand, the lagging strand runs in the opposite direction, heading away from the replication fork, and is synthesized in a series of short fragments known as Okazaki fragments, consequently requiring many primers. The RNA primers of Okazaki fragments are subsequently degraded by RNase H and DNA Polymerase I (exonuclease), and the gaps (or nicks) are filled with deoxyribonucleotides and sealed by the enzyme ligase.
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