Name: dna replication teams in the replisome
Uploaded: 2020-11-23T11:47:35.000Z
Duration: 3 min 1 s
Channel: JoVE

K12 Standards

Alabama Course of Study Science

9-12

Biology

from-molecules-to-organisms-structures-and-processes

From Molecules to Organisms: Structures and Processes

9-12.B.1.3

The Replisome

DNA replication is carried out by a highly coordinated multi-protein assembly known as the DNA replication machinery or the replisome, which increases the efficiency of DNA replication.
The core components of the machinery are helicase, single-strand DNA binding proteins, DNA primase, sliding clamps, a clamp loader, and multiple DNA polymerases, which are all associated with each other near the replication fork.
Replicative DNA polymerases have a processivity of around 10 nucleotides, which is the number of nucleotides it can add to the daughter strand before dissociating from the template strand.&#160;
This is too inefficient to copy entire genomes in a reasonable timeframe and this problem is solved with the help of sliding clamp proteins.
When ATP associates with the clamp loader protein, they will bind to and open the sliding clamp so that its ring-like structure can surround the primer-template DNA complex.&#160; Once bound, the clamp loader hydrolyzes the ATP to ADP, causing the clamp loader to disassociate and the clamp to close around the DNA.
Then, DNA polymerase binds to the clamp proteins and together they slide along the template DNA, tethering DNA polymerase to the strand and increasing its processivity up to 1000 nucleotides.&#160;&#160;
This increased processivity allows the DNA polymerase to carry out continuous DNA replication on the leading strand.
However, on the lagging strand template, another DNA polymerase performs discontinuous DNA replication in a manner that allows the DNA polymerase molecules to synthesize the leading and lagging strands simultaneously. This process is sometimes described as the &#8220;Trombone model.&#8221;
The lagging strand and its template strand form a loop when DNA polymerase initiates Okazaki fragment synthesis from an RNA primer.&#160;
The DNA loop grows from both directions as helicase unwinds DNA and the lagging strand is synthesized.
When the DNA polymerase encounters the next RNA primer, it detaches from the template strand.&#160; Meanwhile, primase adds another primer to the lagging strand, and the growing DNA loop is released.&#160;
The clamp and clamp loader proteins allow DNA polymerase to quickly reassociate with the primed DNA template.
The formation and subsequence collapse of the DNA loop repeats with the synthesis of each new Okazaki fragment.

het replisome

DNA-replicatie wordt uitgevoerd door een groot complex van eiwitten die op een gecoördineerde manier werken om high-fidelity DNA-replicatie te bereiken. Samen staat dit complex bekend als de DNA-replicatiemachinerie of het replisoom.

De synthese van de leidende en achterblijvende strengen is een sterk gecoördineerd proces. Om dit uit te leggen werd in 1980 het ‘Trombone-model’ voorgesteld door Bruce Alberts. De vorming van de DNA-lus begint wanneer een primer wordt gesynthetiseerd op de achterblijvende ouderstreng. De lus groeit met de synthese van een Okazaki-fragment en lost uiteindelijk op wanneer de synthese van het fragment eindigt. Het DNA-polymerase hecht zich vervolgens aan een andere primer en de hele cyclus, van lusvorming tot ineenstorting, wordt herhaald. Met het replisome kunnen alle activiteiten worden gecoördineerd als een replicatiemachinecomplex.

Replisome componenten

Helicasen ontspannen zich en scheiden dubbelstrengs DNA. Deze enzymen zijn aanwezig als een enkele hexameerring in prokaryoten en als een dubbele hexameerring in eukaryoten. Eukaryotische helicasen zijn afhankelijk van extra eiwitten, Cdc45 en GINS, om te kunnen functioneren. Enkelstrengige DNA-bindende (SSB) eiwitten zorgen ervoor dat de DNA-strengen niet opnieuw versmelten. In prokaryoten bestaan SSB's uit een enkele subeenheid, terwijl ze in eukaryoten een heterotrimeer eiwit zijn dat bekend staat als replicatie-eiwit A (RPA).

Primase voegt een RNA-primer toe aan de plek waar de DNA-synthese zal ontstaan. In prokaryoten is primase aanwezig als een enkel subeenheid-enzym genaamd DnaG, dat een RNA-primer van ongeveer 12 nucleotiden synthetiseert. In eukaryoten synthetiseert een multisubunit-enzym, DNA-polymerase-α-primase, een RNA-DNA-hybride primer van ongeveer 25 nucleotiden. Naast polymerase-α zal replicatief polymerase het nieuw gesynthetiseerde DNA uitbreiden. Hoewel een enkel type replicatief polymerase, DNA-polymerase III, aanwezig is in prokaryoten, zijn er twee verschillende typen replicatieve polymerasen, Pol ε en Pol δ, aanwezig in eukaryoten, voor respectievelijk de synthese van de leidende streng en de achterblijvende streng.

Sliding Clamp-eiwitten houden de polymerasen vast aan de DNA-sjabloon. β-clamp, een homodimeer eiwit, fungeert als de klem in prokaryoten. Bij eukaryoten wordt dezelfde taak uitgevoerd door prolifererend celnucleair antigeen (PCNA), een homotrimeer eiwit. De centrale porie van de schuifklem is positief geladen, waardoor deze elektrostatische interacties kan hebben met de negatief geladen fosfaatruggengraat van het DNA. De klem wordt door een klemlader aan het DNA bevestigd. Deze pentamere eiwitten behoren tot de AAA+-klasse van ATPasen. De eukaryote klemlader staat bekend als replicatiefactor C, terwijl de prokaryote E. coli klemlader bekend staat als het γ-complex; Er wordt echter aangenomen dat de klemeiwitten en klemladers evolutionaire homologen zijn, in tegenstelling tot veel andere componenten van het replisoom.

Het Replisome

DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this ...

Education

JoVE Core

Molecular Biology

Moleculaire biologie

Unit 1

DNA Replication

DNA-replicatie

KEY TERMS AND CONCEPTS

BELANGRIJKE TERMEN EN CONCEPTEN

dna replication teams in the replisome

DNA replication depends on the replisome, a coordinated complex of proteins that copies DNA with high accuracy. This DNA replication machinery brings many parts together so the leading and lagging strands can be made at the same time.

A key idea in this process is the trombone model, proposed by Bruce Alberts in 1980. In this model, a loop forms on the lagging strand when a primer is made. The loop grows as an Okazaki fragment is synthesized, then falls apart when that fragment is finished. DNA polymerase then moves to a new primer, and the cycle repeats.

Helicases start the process by unwinding and separating double-stranded DNA. In prokaryotes, helicase is a single hexamer ring, while in eukaryotes it forms a double hexamer ring. Eukaryotic helicases also need the extra proteins Cdc45 and GINS to work. Single-stranded DNA binding proteins keep the separated strands from reannealing. In prokaryotes, these are SSB proteins with one subunit. In eukaryotes, the equivalent protein is replication protein A, or RPA, which is a heterotrimer.

Primase adds the RNA primer that gives DNA synthesis a starting point. In prokaryotes, primase is the single-subunit enzyme DnaG, and it makes an RNA primer of about 12 nucleotides. In eukaryotes, DNA polymerase-alpha primase makes an RNA-DNA hybrid primer of about 25 nucleotides. After that, the replicative polymerase extends the new DNA strand. Prokaryotes use DNA polymerase III, while eukaryotes use Pol epsilon for the leading strand and Pol delta for the lagging strand.

Sliding clamp proteins hold the polymerases on the DNA template so copying can continue efficiently. The prokaryotic beta-clamp is a homodimer, and the eukaryotic clamp is proliferating cell nuclear antigen, or PCNA, which is a homotrimer. The clamp has a positively charged central pore that interacts with the negatively charged DNA backbone. A clamp loader places the clamp onto DNA. These pentameric AAA+ ATPases include the eukaryotic replication factor C and the E. coli gamma complex. The clamp proteins and clamp loaders are thought to be evolutionary homologs, unlike many other replisome components.

DNA Replication Teams in the Replisome

replication in prokaryotes

Prokaryotic DNA Replication Steps

Prokaryotic DNA replication copies the chromosome before a cell divides. In bacteria, this process starts at a specific DNA site called the origin of replication. From there, the cell builds new DNA strands using enzymes that match each base with its partner. The DNA double helix first opens so replication can begin. As the strands separate, each original strand serves as a template. New nucleotides are added in a set order, which helps the cell make an accurate copy of its genetic material. <br...

replication in eukaryotes

Eukaryotic DNA Replication Steps

Eukaryotic DNA replication copies the genome before a cell divides. In eukaryotes, this process happens in a controlled sequence so each new cell can receive genetic information. Replication begins at specific sites on the DNA called origins. The DNA is opened, and each original strand serves as a template, meaning it guides the building of a new complementary strand. This sets up two DNA molecules from one original molecule. The process then continues as enzymes add matching nucleotides to...

dna base pairing

DNA Base Pair Rules

DNA base pairing follows specific rules that help DNA carry genetic information. In DNA, the bases pair in a regular way, with adenine matching thymine and cytosine matching guanine. These pairs fit together because of their chemical shapes and bonding patterns. These base pairs also support accurate DNA replication. When DNA copies itself, each strand can guide the formation of a new partner strand using the pairing rules. Understanding base pairing helps explain how DNA stays organized and...

the dna replication fork

DNA Replication Stress and Fork Stalling

DNA replication stress and fork stalling can disrupt the copying of a cell’s genome. The replication fork is the Y-shaped site where DNA strands separate and new complementary strands are made at the same time. This linked unwinding and synthesis helps cells duplicate DNA efficiently and with fewer errors. In organisms with small circular DNA, such as E. coli, replication often begins at a single origin. That setup creates two replication forks that move in opposite directions from the opening...

proofreading

Proofreading in DNA Replication

Proofreading in DNA replication helps keep DNA copying accurate. It is a built-in check that reduces mistakes as new DNA is made. DNA polymerase is the enzyme that carries out this checking during replication. When it adds a wrong nucleotide, it can remove the error and replace it with the correct one. This correction step helps maintain the correct DNA sequence. Proofreading is an important part of DNA replication because it supports genetic accuracy. By catching mistakes as they happen, the...

dna helicases

Helicase Motor Proteins in DNA Unwinding

Helicase motor proteins help unwind DNA during key cell processes. They use energy from ATP hydrolysis, which is the breakdown of ATP to release usable energy. This movement lets helicases separate DNA strands when the cell needs access to the genetic code. These enzymes are involved in DNA replication, repair, recombination, and transcription. Helicases are found in all living organisms, but their structure, function, and movement can differ. In prokaryotes, the DnaB helicase moves along the...

the replisome

DNA replication depends on the replisome, a coordinated complex of proteins that copies DNA with high accuracy. This DNA replication machinery brings many parts together so the leading and lagging strands can be made at the same time. A key idea in this process is the trombone model, proposed by Bruce Alberts in 1980. In this model, a loop forms on the lagging strand when a primer is made. The loop grows as an Okazaki fragment is synthesized, then falls apart when that fragment is finished.

lagging strand synthesis

Okazaki Fragments in DNA Replication

DNA replication uses a leading strand and a lagging strand, and they are made at different rates. The leading strand starts first. The lagging strand starts later, moves more slowly, and is made in pieces. The two strands also differ in direction and starting steps. Leading strand synthesis follows the opening replication fork. Lagging strand synthesis goes in the opposite direction. The leading strand needs one RNA primer, while the lagging strand needs multiple RNA primers. After priming,...

mismatch repair

Fixing DNA Copying Errors

Mismatch repair helps fix DNA copying errors after replication. When DNA is copied, small mistakes can slip through. This repair system finds those mismatches and corrects them before they become permanent changes. The process depends on a group of mutator proteins. These proteins work together to detect the error, remove the incorrect section, and help replace it with the proper DNA sequence. In this way, mismatch repair supports the accuracy of genetic information. If the repair system does...

dna topoisomerases

How Topoisomerases Untwist DNA

DNA topoisomerases are enzymes that untwist DNA when it becomes overcoiled during cell processes such as DNA replication and transcription. They help regulate positive and negative DNA supercoiling without changing the nucleotide sequence. Clockwise overwinding creates positively supercoiled DNA, while counterclockwise underwinding produces negatively supercoiled DNA. Topoisomerases are grouped into two main types. Type I topoisomerases act on one strand of double-stranded DNA, and they...

telomeres and telomerase

How Telomerase Protects Chromosome Ends

Telomerase helps protect chromosome ends during eukaryotic DNA replication. When the final primer is removed, a single-stranded DNA fragment can remain at the end of the chromosome. That piece cannot be copied in the same way as the rest of the strand because there is no 3' end for new DNA to attach to. If this end piece is not replaced, chromosomal DNA is gradually lost each time a cell divides. The exposed single-stranded DNA can also trigger a DNA damage response from enzymes that detect it.

non-nuclear inheritance

Cytoplasmic Inheritance in Cells

Cytoplasmic inheritance explains how some traits are passed through cell structures outside the nucleus. These traits come from organelles such as mitochondria and chloroplasts. They do not follow the usual nuclear pattern of inheritance. Mitochondria and chloroplasts each contain their own DNA. Because of this, they can carry genetic information that affects cell function and certain visible traits. In high school biology, this topic helps explain why not all inherited traits come from...

animal mitochondrial genetics

Animal Mitochondrial DNA and Inheritance

Animal mitochondrial DNA has its own genome, separate from nuclear DNA. It is a double-stranded, closed circular molecule with about 20,000 base pairs. One strand is the heavy, or H-strand, because it is rich in guanine. The other is the light, or L-strand, because it is rich in cytosine. Compared with nuclear DNA, mitochondrial DNA has very little non-coding sequence. It also has no introns. Its genes are packed closely together, and some even overlap. The D-loop is the main non-coding...

comparing mitochondrial, chloroplast, and prokaryotic genomes

comparing-mitochondrial-chloroplast-and-prokaryotic-genomes

How Organelle Genomes Resemble Bacteria

Mitochondrial and chloroplast genomes still keep several traits from their bacterial ancestors. They also show changes that happened after these organelles evolved inside eukaryotic cells. Eukaryotic cells are cells with a nucleus, while prokaryotes are cells without one. Like prokaryotic genomes, mitochondrial and chloroplast genomes do not bind with histone-like proteins. They also do not show the complex chromosome-like packaging seen in eukaryotes. Mitochondria and chloroplasts divide by...

export of mitochondrial and chloroplast genes

export-of-mitochondrial-and-chloroplast-genes

How Organelles Pass Genes to the Nucleus

Eukaryotic cells can carry three genetic systems: nuclear, mitochondrial, and chloroplast. Over time, many genes from mitochondria and chloroplasts have moved into the nucleus. In some plant species, this gene transfer is still happening. This movement has affected large parts of the genome in some organisms. About 18% of the Arabidopsis thaliana nuclear genome is thought to come from the chloroplast's cyanobacterial ancestor. In yeast, around 75% of the genome is derived from the...

single-strand dna binding proteins

How SSB Proteins Protect DNA Strands

Single-strand DNA-binding proteins, or SSB proteins, help protect DNA during replication. When double-stranded DNA is unwound, the two strands separate. SSB proteins bind to the exposed single strands and keep them stable. These proteins bind in a sequence-independent manner. That means the order of the nitrogenous bases does not have to follow a specific pattern for SSB proteins to attach. Their binding straightens single-stranded DNA and makes it more rigid. This support may help DNA...

key terms and concepts

Explore how the replisome coordinates DNA replication, from helicase and primase to polymerases, sliding clamps, and the trombone model.

DNA Replication Teams in the Replisome | JoVE K12

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9-12.B.1.2.a

DNA Replication Teams in the Replisome

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