DNA Replication

DNA ligase

- After the RNA primer is replaced with DNA and the replacement is completed, the adjacent Okazaki fragments are joined (ligated) by an enzyme called DNA ligase.

Fourth Step in DNA Replication

- Antiparallel elongation - DNA Polymerase III (DNAP III) attaches to each primer on the parental strand and moves in the 3' to 5' direction. - As it moves, it adds nucleotides to the new strand in the 5' to 3' direction. - Because the 2 DNA strands in a double helix are antiparallel and a new DNA strand can be synthesized only at the 3' end, the 2 daughter strands are synthesized in quite different ways. One of the parental strands has a left-to-right 5'-to-3' orientation, whereas the other parental strand runs in the opposite direction, with a left-to-right 3'-to-5' orientation - The DNA polymerase/DNAP III that follows helicase is known as the leading strand and it only requires one primer - The DNA polymerase/DNAP III on the other parental strand that moves away from helicase is known as the lagging strand and requires many primers

Base pairing during DNA replication

- As the two strands of DNA separate, each of the parental strands serves as a template, or model, for the synthesis of a complementary daughter strand. The sequence of bases along the parental strand determines the sequence of bases along the daughter strand because wherever one strand carries an A, the other must carry at T, and wherever one carries a G, the other must carry a C. - As a result, each of the daughter DNA molecules is identical in sequence to the parental DNA molecules--except possibly for rare errors called mutations.

Copying DNA: semi-conservative

- Base pairing allows each strand to serve as a template for a new strand - New strand is 1/2 parent template and 1/2 new DNA - Significance of this type of DNA replication: this genetic information is ensured to be transferred from one generation to the next generation with a high fidelity. (fidelity = ability to accurately replicate a template)

Semi-conservative model

- By analyzing samples of DNA after each generation, it was found that the parental strands were following the semi-conservative model. - The two parental strands each make a copy of itself - After one round of replication, the two daughter molecules each have one parental strand and one new strand.

Leading strand

- DNA Polymerase III moves 3' to 5' following helicase - The replication fork is moving to the left. As the replication fork moves along, it creates a region of single-stranded DNA. An RNA primer is first laid down, and then, DNA polymerase can take over. - When one daughter strand that is being synthesized using the other strand as a template has its 3' end pointed toward the replication fork, the parental double helix unwinds, and nucleotides can be added onto the 3' end. As a result, this daughter strand can be synthesized as one long, continuous polymer.

DNA polymerase

- DNA is replicated by an enzyme called DNA polymerase. - This enzyme is a component of a large protein complex that makes copies of DNA and therefore carries out DNA replication. DNA polymerases exist in all organisms and are highly conserved, meaning that they vary little from one species to another because they carry out an essential function. 2 main properties: - It can only attach one nucleotide to another nucleotide (in other words, it can only elongate the end of an existing piece of DNA or RNA and cannot lay down the first nucleotide of a newly synthesized strand on its own. - As a result, each new DNA strand must begin with a short stretch of RNA that serves as a primer, or starter, for DNA synthesis. The primer is made by an enzyme called RNA primase, which synthesizes a short piece of. RNA complementary to the DNA parental strand. Once the RNA primer has been synthesized, DNA polymerase extends it, adding successive DNA nucleotides to the 3' end of the growing strand. - It can only add nucleotides to the 3' end of another nucleotide, not the 5' end. DNA synthesis occurs when the 3' hydroxyl group attacks the phosphate group of an incoming nucleotide triphosphate, so as a result, DNA synthesis can only occur in the 5' -to-3' direction. As the incoming nucleotide triphosphate is added to the growing DNA strand, one of the nucleotide's high-energy phosphate bonds is broken, providing energy for the reaction. The outermost two phosphates are released in the process.

Lagging strand

- DNA polymerase III moves 3' to 5' away from helicase - As the other strand has a left-to-right 3'-to-5' orientation, the daughter strand has a left-to-right 5'-to-3' orientation. - As a result, the 5' end of the daughter strand is pointed toward the replication fork. However, the daughter strand cannot grow in that direction because DAN polymerase can add nucleotides only to the 3' end of a growing strand. To enable synthesis of the top daughter strand, the replication fork moving to the left first creates a single-stranded region of parental DNA ranging in length from a few hundred to a few thousand nucleotides. An RNA primer is laid down. Synthesis of the new strand occurs at its 3' end as usual, which means that the daughter strand grows in a direction away from the replication fork,. The result is that the top daughter strand is synthesized in relatively short, discontinuous pieces called Okazaki fragments. As the parental double helix unwinds, a new piece is initiated at intervals, and each new piece is elongated at its 3' end until it reaches the piece in front of it.

DNA replication

- DNA replicates during the S phase of the cell cycle

First Step in DNA Replication

- DNA replication begins at sites called origins of replication - Various proteins attach to the origin of replication and open the DNA to form a replication fork - The replication fork is like a fork in the road, with a singel road splitting into two roads going off in different dieections. - Large team of enzymes coordinates replication. More than a dozen enzymes and other proteins participate in DNA replication.

RNA primer and leading/lagging strands

- For both the leading and lagging strands, each new DNA strand begins with a short stretch of RNA. Befaeuse DNA polymerase extends an RNA primer, all new DNA strands have a short stretch of RNA at their 5' end. - For the lagging strand, there are many such primers, one for each of the discontinuous Okazaki fragments of newly synthesized DNA. As each of these fragments is elongated by DNA polymerase, it grows toward the primer of the fragment in front of it. When the growing fragment comes into contact with the primer of the fragment synthesized earlier, a different DNA polymerase takes over, removing the earlier RNA primer and extending the growing fragment with DNA nucleotides to fill the space left by its removal

Second Step in DNA Replication

- Helicase will unwind the DNA strands at each replication fork (more than one) by breaking hydrogen bonds holding the base pairs together. - To keep the DNA from re-bonding with itself, proteins called single strand binding proteins (SSBPs) bind to the DNA to keep it open - Topoisomerase will help prevent strain ahead of the replication fork by relaxing supercoiling. It works upstream from the replication fork to relieve the stress that results from unwinding the double helix at the replication fork. Topoisomerases are a family of enzymes that wind or unwind DNA to help relieve stress that occurs during both replication and transcription.

Meselson and Stahl experiment

- In 1954, Meselson and Stahl performed an experiment using bacteria. - They reasoned that if they were a way to distinguish newly synthesized daughter DNA strands ("new strands") from previously synthesized parental strands ("old strands"), the products of replication could be observed and the mechanism of replication determined. Process: 1. Bacteria was cultured with a heavy isotope--15N. 2. Bacteria was transferred to a medium with 14N, a light isotope. 3. DNA was centrifuged and analyzed after each replication. - Demonstrated that semi-conservative replication occurs in bacteria

The role of enzymes in DNA replication

- Many enzymes play roles in DNA replication. - Helicase unwinds the parental strands at the replication fork - Single-stranded binding proteins help to stabilize single strands of DNA - Topoisomerase relieves the stress that results from winding the double helix. DNA polymerase synthesizes new DNA strands.

Proofreading

- Most DNA polymerases are capable of proofreading--a process in which a DNA polymerase can immediately correct its own errors. When each new nucleotide comes into line in preparation for attachment to the growing DNA strand, the nucleotide is temporarily held in place by hydrogen bonds that form between the base in the new nucleotide and the base across the way in the parental strand. The strand being synthesized and the parental strand therefore have complementary bases--A paired with T, or G, or G paired with C. - However, on rare occasions, an incorrect nucleotide is attached to the new DNA strand. When this happens, DNA polymerase can correct the error because it detects mispairing between the template and the most recently added nucleotide. After it detects mispairing between a base in the parental strand and an newly added base in the daughter strand, it removes the incorrect nucleotide and inserts the correct one in its place. - Mutations resulting from errors in nucleotide incorporation still occur, but proofreading reduces their number. For example, in E. coli, about 99% of the incorrect nucletoides that are incorporated during replication are removed and repaired by the proofreading function of DNA polymerase. Those that slip past proofreading and other repair systems lead to mutations, which are then copied and passed on to daughter cells. - Some of these mutations may be harmful, but others are neutral and a rare few may be benifical. These mutations are the ultimate source of genetic variation that we see among individuals of the same species and among species. They are essential in the process of evolution because they allow populations of organisms to change through time and adapt to their environment.

Bidirectional replication

- Once the DNA is opened at the origin, two replication forks are formed spontaneously. - These two replication forks move in opposite directions as the syntheses continue.

Why is accurately reproducing DNA and the sequence of nucleotides as precisely as possible for?

- Reproducing the sequence of nucleotides as precisely as possible is important because mistakes that go unrepaired may be harmful to the cell or organism. - An unrepaired error in DNA replication results in a mutation, which is a change in the genetic information in DNA. For example, a mutation in DNA can cause the genetic difference between virulent and nonvirulent bacteria.

Key characteristics of replication

- Semi-conservative replication - Bidirectional replication - Semi-continuous replication - High fidelity

Third Step in DNA Replication

- The enzyme primase initiates replication by adding short segments of RNA, called primers, to the parental DNA strand. - Primers serve as the foundation for DNA synthesis -The enzymes that synthesize DNA can only. attach new DNA nucleotides to an existing strand of nucleotides

Fifth Step in DNA Replication

- The leading strand is synthesized in one continuous segment, but since the lagging strand moves away from the replication fork it is synthesized in chunks - Okazaki fragments: segments of the lagging strand - The presence of leading and lagging strands during DNA replication is a consequence of the antiparallel nature of the 2 strands in ad DNA double helix and the fact that DNA polymerase can synthesize DNA only in the 5'-to-3' direction. Nevertheless, the copying mechanism ins the same on both strands: after the two strands of parental DNA separate, each serves as a template for the synthesis of a daughter strand according to the base-pairing rules of A-T and G-C, with each successive nucleotide being adde to the 3' end of the growing strand.

Models of DNA replication

- There were 3 alternative models for DNA replication: - Conservative model: parental strands are fully conserved. The original DNA molecule remains intact and the daughter DNA molecule is completely new. - Semi-conservative model: each parental strand makes a copy of itself. After 1 round of replication, each molecule has 1 parental new strand. - Dispersive model: random mix of parental and daughter DNA *Semi-conservative model was the correct model

How does DNA polymerase do proofreading?

- When an incorrect nucleotide is added, the proofreading function of DNA polymerase removes the incorrect nucleotide. - Then, the correct nucleotide is added to replace the incorrect one. - DNA polymerase has a proofreading function, allowing it to identify and replace an incorrect nucleotide during DNA replication.

Replication of prokaryotes

-The replication process starts from the origin, and proceeds in two opposite directions. It is named theta (θ) replication.

A summary of DNA replication

1. Helicases unwind the parental double helix 2. Single-strand binding proteins stabilize the unwound parental DNA 3. The leading strand is synthesized continuously in the 5' --> 3' direction by DNA polymerase. 4. The lagging strand is synthesized discontinuously. Primase synthesizes a short RNA primer, which is extended by DNA polymerase to form an Okazaki fragment. 5. After the RNA primer is replaced by DNA (by another DNA polymerase), DNA ligase joins the Okazaki fragment to the growing strand.

Replication of eukaryotes

Chromosomes of eukaryotes have multiple origins - Origins of DNA replication (around 100 kilobases/kb). This amounts to 30,00 DNA replication origins per cell. - 30,000 DNA replication origins are activated with each cell division. At each of these origins, spaced approximately 100 kb apart, protein complexes form that prepare the DNA to initiate bidirectional replication, that is, fire. - The space between two adjacent origins is called the replicon, a functional unit of replication.