Name: dna copying: leading and lagging strands
Uploaded: 2020-09-24T15:06:53.000Z
Duration: 2 min 40 s
Channel: JoVE

DNA replication occurs by synthesizing new strands of DNA using existing strands as a template. The two new double helices each contain an original template strand and a newly synthesized daughter strand, which is why this process is known as semiconservative replication.&#160;&#160;
To begin replication, an enzyme, helicase, unwinds the DNA helix and breaks the hydrogen bonds between the two strands. It then separates the individual strands forming a Y-shaped structure, known as the replication fork, where the template strands can be accessed by additional enzymes.
Another enzyme, primase, adds short RNA fragments called primers onto each template strand. These primers are essential for the synthesis of DNA as DNA polymerase can only add nucleotides to an existing strand.&#160;
DNA polymerase adds to the growing daughter strands on both template DNA strands. The addition of nucleotides is guided by the sequence of the original DNA strand according to the DNA pairing rules.
Synthesis of one of the daughter strands, the leading strand, occurs in the direction of the replication fork movement. The other strand, the lagging strand, is synthesized in the opposite direction.
This leads to the leading strand being synthesized as a continuous polymer, whereas the lagging strand is synthesized as short fragments. This occurs because DNA can be synthesized only in the 5&#8217; to 3&#8217; direction.
Before their addition to the growing polymer, nucleotides exist as deoxyribonucleoside triphosphates, with three phosphates attached to the fifth carbon on the sugar.
This free nucleotide triphosphate reacts with the 3&#8217; hydroxyl groups. This is the OH that is attached to the third carbon of the sugar at the end of the growing strand.&#160; The reaction causes the release of pyrophosphate and the formation of a phosphodiester bond between the two nucleotides.
After the synthesis of the new strands, RNase H or additional variants of DNA polymerase remove the primers and synthesize DNA in their place. The gaps between the fragments are then sealed by DNA ligase to generate a continuous strand.
The addition of nucleotides continues until two replication forks meet each other, resulting in the completed replication.

dna replication

DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.&#160; After replication, each double-stranded DNA includes one parental or &#8220;old&#8221; strand and one &#8220;new&#8221; strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Replication in Prokaryotes
DNA replication uses a large number of proteins and enzymes. The enzyme helicase separates the two strands of DNA. As helicase moves along the DNA, it separates the two strands to form a Y-shaped structure known as the replication fork. Following this, the enzyme primase adds short stretches of RNA known as primers to initiate the synthesis of DNA by DNA polymerase, the enzyme responsible for DNA synthesis. In bacteria, three main types of DNA polymerases are known: DNA pol I, DNA pol II, and DNA pol III. DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair. DNA pol III adds deoxyribonucleotides, each complementary to a nucleotide on the template strand, one by one to the 3&#8217;-OH group of the growing DNA chain. DNA polymerase III can only extend in the 5&#8217; to 3&#8217; direction. The addition of these nucleotides requires energy. This energy is present in the bonds of three phosphate groups attached to each nucleotide, similar to how energy is stored in the phosphate bonds of adenosine triphosphate (ATP). When the bond between the phosphates is broken and diphosphate is released, the energy released allows for the formation of a covalent phosphodiester bond by dehydration synthesis.
The DNA double helix is antiparallel; that is, one strand is oriented in the 5&#8217; to 3&#8217; direction and the other is oriented in the 3&#8217; to 5&#8217; direction. During replication, one strand, which is complementary to the 3&#8217; to 5&#8217; parental DNA strand, is synthesized continuously toward the replication fork because polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand. The other strand, complementary to the 5&#8217; to 3&#8217; parental DNA, grows away from the replication fork, so the polymerase must move back toward the replication fork to begin adding bases to a new primer, again in the direction away from the replication fork. It does so until it bumps into the previously synthesized strand, and then it moves back again. These steps produce small DNA sequence fragments known as Okazaki fragments, each separated by RNA primer. The strand with the Okazaki fragments is known as the lagging strand, and its synthesis is said to be discontinuous.
After the synthesis by DNA polymerase III, the primers are removed by the exonuclease activity of DNA polymerase I, and the gaps are filled in. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the formation of a covalent phosphodiester linkage between the 3&#8217;-OH end of one DNA fragment and the 5&#8217; phosphate end of the other fragment, stabilizing the sugar-phosphate backbone of the DNA molecule.
Replication in Eukaryotes
Eukaryotic genomes are much more complex and larger than prokaryotic genomes and are typically composed of multiple linear chromosomes. The human genome, for example, has 3 billion base pairs per haploid set of chromosomes, and 6 billion base pairs are inserted during replication. There are multiple origins of replication on each eukaryotic chromosome; the human genome has 30,000 to 50,000 origins of replication. The rate of replication is approximately 100 nucleotides per second&#8212;10 times slower than prokaryotic replication.
The essential steps of replication in eukaryotes are the same as in prokaryotes. Before replication can start, the DNA has to be made available as a template. Eukaryotic DNA is highly supercoiled and packaged, which is facilitated by many proteins, including histones. Following initiation of replication, in a process similar to that found in prokaryotes, elongation is facilitated by eukaryotic DNA polymerases. The leading strand is continuously synthesized by the eukaryotic polymerase enzyme pol &#948;, while the lagging strand is synthesized by pol &#949;. The enzyme ribonuclease H (RNase H), instead of a DNA polymerase as in bacteria, removes the RNA primer, which is then replaced with DNA nucleotides. The gaps that remain are sealed by DNA ligase.
As in prokaryotes, the eukaryotic DNA polymerase can add nucleotides only in the 5&#8217; to 3&#8217; direction. In the leading strand, synthesis continues until it reaches either the end of the chromosome or another replication fork progressing in the opposite direction. On the lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer. When the replication fork reaches the end of the linear chromosome, there is no place to make a primer for the DNA fragment to be copied at the end of the chromosome. These ends thus remain unpaired, and, over time, they may get progressively shorter as cells continue to divide.
This text is adapted from <a href="https://openstax.org/books/microbiology/pages/11-2-dna-replication">Openstax, Microbiology, Chapter 11.2: DNA Replication</a>.

DNA Replication

DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary ...

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dna copying: leading and lagging strands

DNA replication copies genetic material before a cell divides. Each original DNA strand serves as a template for a new complementary strand. The result is semiconservative replication, which means each new DNA molecule contains one old strand and one new strand. The two daughter cells then receive DNA with the same sequence.

Replication starts when helicase separates the two strands of the double helix. This creates a Y-shaped replication fork. Primase then adds short RNA primers that give DNA polymerase a place to begin DNA synthesis. In bacteria, the main DNA polymerases are DNA pol I, DNA pol II, and DNA pol III. DNA pol III carries out DNA synthesis, while DNA pol I and DNA pol II mainly help with repair.

DNA pol III adds deoxyribonucleotides one at a time to the 3'-OH end of the growing strand. It can extend DNA only in the 5' to 3' direction. The energy for this process comes from the three phosphate groups on each nucleotide. When a phosphate bond is broken and diphosphate is released, that energy helps form the phosphodiester bond in the DNA backbone.

Because the DNA double helix is antiparallel, the two template strands face opposite directions. The strand copied toward the replication fork is made continuously and is called the leading strand. The other strand is copied away from the fork. It must start over with new primers as the fork opens, so it is made in short stretches called Okazaki fragments. This strand is the lagging strand, and its synthesis is discontinuous.

After DNA pol III finishes, DNA pol I removes the RNA primers in bacteria, and the gaps are filled in. DNA ligase then seals the remaining nicks by forming covalent phosphodiester bonds between adjacent DNA fragments. This step strengthens the sugar-phosphate backbone of the new DNA molecule.

Eukaryotic DNA replication follows the same basic steps, but the genome is larger and more complex. Eukaryotic chromosomes are linear and have many origins of replication. In humans, the genome has 3 billion base pairs per haploid set, and about 6 billion base pairs are copied during replication. Human chromosomes also have about 30,000 to 50,000 origins of replication, and replication occurs at about 100 nucleotides per second, which is much slower than in prokaryotes.

Before replication begins, eukaryotic DNA must be made available as a template. It is tightly packed and supercoiled, and histones help organize this packaging. After initiation, eukaryotic DNA polymerases carry out elongation. Polymerase delta (pol δ) synthesizes the leading strand, and polymerase epsilon (pol ε) synthesizes the lagging strand. RNase H removes the RNA primers, DNA nucleotides replace them, and DNA ligase seals the final gaps. Because eukaryotic DNA polymerase also works only in the 5' to 3' direction, the lagging strand is still made in short pieces. Near the ends of linear chromosomes, there is no place to add a final primer, so these ends remain unpaired and can become shorter over time as cells keep dividing.

DNA Copying: Leading and Lagging Strands

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Explore how DNA replication uses leading and lagging strands, RNA primers, DNA polymerase, ligase, and semiconservative copying in bacteria and eukaryotes.

DNA Copying: Leading and Lagging Strands | JoVE K12

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DNA Copying: Leading and Lagging Strands

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