Name: dna polymerase error correction
Uploaded: 2023-04-30T11:43:46.000Z
Duration: 1 min 31 s
Channel: JoVE

K12 Standards

north-carolina-k-12-science-standard-course-of-study

NC K-12 Science Standard Course of Study

9-12

Biology

heredity-inheritance-and-variation-of-traits

Heredity- Inheritance and Variation of Traits

LS.Bio.6.2

Proofreading

During DNA replication, complementary base pairing dictates the sequence of nucleotides added to the new strand &#8212; adenine pairs with thymine and guanine pairs with cytosine.
However, nucleotides can sometimes be incorrectly paired, for instance, adenine with cytosine. Such errors are prevented or repaired by the proofreading properties of DNA polymerases carried out during DNA synthesis.
First, DNA polymerase has a high affinity for complementary nucleotides, which improves the accuracy of selecting the correct incoming nucleotide to pair with the template strand.
Second, as the nucleotides start pairing, the DNA polymerase undergoes a conformational change that makes the mispaired nucleotides more likely to dissociate but retains the correctly paired nucleotides.
Third, if an incorrect nucleotide is somehow added to the growing 3&#39; end, the structural deviation caused by this incorrect pairing causes the DNA polymerase to pause.
In a process called exonucleolytic proofreading, the 3&#39; end is then transferred to the exonuclease site of the DNA polymerase. The polymerase subsequently removes the incorrect nucleotide in a 3&#39; to 5&#39; direction, replaces it with the correct nucleotide, and resumes DNA synthesis.

proofreading

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template DNA strand. DNA polymerase has a higher affinity to add the correct base and ensures fidelity during DNA replication. Furthermore,&#160; it exhibits proofreading activity during replication, using an exonuclease domain that cuts off incorrect nucleotides from the nascent DNA strand.
Errors During Replication are Corrected by the DNA Polymerase Enzyme
Genomic DNA is synthesized in the 5&#8217; to 3&#8217; direction. Each cell contains a number of DNA polymerases that play different roles in synthesizing and correcting mistakes in the DNA. For example, DNA polymerase delta and epsilon possess proofreading ability when replicating nuclear DNA. These polymerases &#8220;read&#8221; each base after it is added to the new strand. If the newly-added base is incorrect, the polymerase reverses direction (moving from 3&#8217; to 5&#8217;) and uses an exonucleolytic domain to cut off the incorrect base. Subsequently, the excised base is replaced with the correct base.
Mutations in the Exonuclease Domain of DNA Polymerase are Linked to Cancers
Proofreading is important for preventing mutations from occurring in newly-synthesized DNA, but what happens when the proofreading mechanism fails? When a mutation alters the exonuclease domain of DNA polymerase, it loses the ability to remove incorrect nucleotides. In consequence, mutations can accumulate rapidly throughout the genome. This type of mutation has been linked to various types of cancer.
Low-fidelity DNA Polymerase can Generate Mutated DNA Sequences
Modified DNA polymerases are used in laboratory science for polymerase chain reaction (PCR), an in vitro technique for making many copies of specific fragments of DNA. While high-fidelity polymerases are used when it is important that the end product is perfect, some techniques, such as error-prone PCR, seek to generate mutations in a stretch of DNA on purpose. These techniques use polymerases that have compromised proofreading ability.

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template ...

Education

JoVE Core

Cell Biology

Unit 1

DNA Replication and Repair

KEY TERMS AND CONCEPTS

dna polymerase error correction

DNA polymerase uses proofreading to help correct errors during DNA replication. As it builds the daughter strand, the enzyme adds nucleotides that match the template DNA strand. This matching helps keep DNA copying accurate and supports replication fidelity.

DNA is made in the 5' to 3' direction, and several DNA polymerases help with this process. In nuclear DNA replication, DNA polymerase delta and DNA polymerase epsilon have proofreading ability. After a new base is added, these polymerases check it. If the base is wrong, the polymerase moves backward from 3' to 5' and uses an exonuclease domain to remove the incorrect nucleotide. The correct base is then added in its place.

Proofreading matters because it helps prevent mutations in newly synthesized DNA. When a mutation changes the exonuclease domain of DNA polymerase, the enzyme can no longer remove wrong nucleotides well. As a result, mutations can build up quickly across the genome. This kind of defect has been linked to several cancers.

Modified DNA polymerases also have a role in the lab. Scientists use them in polymerase chain reaction, or PCR, to make many copies of specific DNA fragments. High-fidelity polymerases are chosen when an accurate product is needed. In contrast, error-prone PCR uses polymerases with reduced proofreading so mutations can be generated on purpose in a DNA sequence.

DNA Polymerase Error Correction

base-pairing and dna repair

DNA Pairing Rules in Repair

DNA pairing rules help guide DNA repair. In DNA, bases pair in a specific way, and those matching pairs support accurate copying and fixing of the molecule. When DNA is damaged or copied incorrectly, repair systems can use the normal base-pairing pattern as a reference. This helps cells find mismatched bases and restore the correct sequence. The process depends on the idea that each base has a preferred partner. Understanding base-pairing is important because it explains how cells protect...

the dna replication fork

DNA Replication Fork in Action

The DNA replication fork is the active region where DNA copies itself. It forms when the two strands of the double helix separate so each strand can serve as a template for a new partner strand. At the fork, the copying process moves in a set direction. The two strands are built in different ways as replication continues, so the fork helps organize how the new DNA is made. The replication fork is a key part of accurate DNA duplication. It shows how the cell opens the double helix, copies the...

lagging strand synthesis

Okazaki Fragments on the Lagging Strand

Lagging strand synthesis builds new DNA in short pieces called Okazaki fragments. This happens during DNA replication because the lagging strand runs in the opposite direction from the direction the replication machinery moves. As the DNA double helix opens, DNA polymerase adds nucleotides only in the 5' to 3' direction. On the lagging strand, this forces the cell to copy the template in segments instead of one continuous stretch. Each segment begins near the replication fork and extends away...

the replisome

Replisome Components in DNA Copying

The replisome is the protein machine that copies DNA during cell division. It works at the replication fork, where the double helix opens so new strands can be made. DNA polymerase is the core enzyme in the replisome. It adds nucleotides to the growing DNA strand. In bacteria, two DNA polymerases work together at the fork. These enzymes are linked to the helicase by a clamp-like protein so they stay attached to the DNA. Helicase unwinds the DNA ahead of the fork. Single-stranded binding...

DNA polymerase uses proofreading to help correct errors during DNA replication. As it builds the daughter strand, the enzyme adds nucleotides that match the template DNA strand. This matching helps keep DNA copying accurate and supports replication fidelity. DNA is made in the 5' to 3' direction, and several DNA polymerases help with this process. In nuclear DNA replication, DNA polymerase delta and DNA polymerase epsilon have proofreading ability. After a new base is added, these polymerases...

replication in prokaryotes

How Prokaryotic DNA Copies Itself

Prokaryotic DNA copies itself through a set of coordinated steps called initiation, elongation, and termination. In bacteria, replication starts when initiator proteins bind to a single origin of replication, or ori, on the circular chromosome. The DNA is then copied in both directions until two DNA molecules are made. Several proteins help open and stabilize the DNA during replication. Topoisomerase cuts one side of the double-stranded phosphate-sugar backbone so the helix can unwind faster.

replication in eukaryotes

Eukaryotic DNA Replication Steps

Eukaryotic DNA replication copies the genome before cell division. It happens in a tightly regulated way so each daughter cell gets a complete set of chromosomes in the nucleus. The process has three stages: initiation, elongation, and termination. Eukaryotic cells use many proteins to manage replication because their chromosomes are linear and their genomes are larger than those of prokaryotes. Replication begins at multiple origins of replication along each chromosome. Initiator proteins...

telomeres and telomerase

Telomeres and Telomerase in DNA Replication

Telomeres and telomerase help protect chromosomes during DNA replication. Telomeres are repeated DNA sequences at chromosome ends, and telomerase is the enzyme that can extend them. Together, they help keep important genetic information from being lost when cells divide. Each time a chromosome is copied, the very end of the DNA strand can be difficult to replicate fully. Telomeres act like a buffer zone at the tips of chromosomes. Telomerase adds new repeats to these ends, which helps maintain...

overview of dna repair

DNA Repair and Genetic Recombination

DNA repair and genetic recombination help cells respond to damage in DNA. DNA can be harmed by normal cell activity or by outside factors. When damage is not fixed, it can affect how genetic information is copied and used. Cells use repair pathways to detect and correct many kinds of DNA damage. Some repair steps remove damaged bases or replace missing pieces of DNA. Other pathways help keep the DNA sequence stable after damage has occurred. Genetic recombination is also part of this process.

base excision repair

Base Excision Repair Pathway

Base excision repair is a DNA repair pathway that fixes small base damage. It helps cells correct changes that can appear in DNA before they cause larger problems. In this pathway, a damaged base is removed first. The cell then processes the site and replaces the missing part with the correct DNA building block. This repair step helps keep genetic information accurate. Base excision repair is one of the main ways cells handle everyday DNA damage. It is an important part of maintaining DNA...

nucleotide excision repair

UV Damage and DNA Repair by NER

UV damage can bend DNA and block replication or transcription. Cells often face mutagens, which are outside factors that damage DNA and can create mutations. If these changes are not repaired, they can lead to disease or cancer, depending on which DNA sequences are affected. Nucleotide excision repair, or NER, is the pathway that helps cells find and remove damaged DNA. In prokaryotes, three proteins carry out this work: UvrA, UvrB, and UvrC. UvrA and UvrB move along the DNA together and look...

mismatch repair

DNA Mismatch Repair and Cancer Risk

DNA mismatch repair fixes copying errors that happen during DNA replication. It helps cells find the wrong nucleotide, remove it, and replace it with the correct one before the cell divides. Human cells carry more than 3 billion base pairs of DNA, so replication must be very accurate. Even with DNA polymerase proofreading, a copying error can still occur about once every 1 million base pairs. One common error is a mismatch, such as A paired with G or T paired with C. The Mutator protein family...

dna damage can stall the cell cycle

DNA Repair Checkpoints in the Cell Cycle

DNA repair checkpoints help cells pause the cell cycle after DNA damage. This pause gives the cell time to check for breaks and repair them before division continues. Cells must do this at critical stages of the cycle. The amount of DNA in the cell depends on when the pause happens. If the cycle stops before DNA replication, the cells will contain twice the normal amount of DNA. If cells stop after DNA replication but before mitosis, they will contain four times the normal amount. Cells use...

fixing double-strand breaks

Repairing DNA Double-Strand Breaks

Repairing DNA double-strand breaks is an important part of DNA maintenance. A double-strand break happens when both strands of the DNA molecule are cut. Cells must fix this damage to keep genetic information stable. The repair process can happen by more than one pathway. Some breaks are repaired by direct joining of the broken ends. Other breaks are repaired by using a matching DNA sequence as a template. These pathways help cells respond to damage in different ways. This topic is closely...

homologous recombination

Homologous Recombination in DNA Repair

Homologous recombination is a DNA repair process that uses a matching DNA sequence as a template. It helps cells fix breaks in DNA and keep genetic information accurate. The process begins when a broken DNA strand finds a similar, or homologous, piece of DNA. That matching sequence guides repair and allows the damaged DNA to be restored. Because the repair uses a copied sequence, the final DNA can closely match the original information. Homologous recombination is also important in genetic...

gene conversion

Gene Conversion During DNA Repair

Gene conversion is a DNA repair event linked to genetic recombination. It can change the sequence of one DNA region by using a related DNA strand as a template. This process is important for understanding how cells repair damage and reshuffle genetic information. During recombination, gene conversion can copy information from one DNA molecule to another. The result is not a simple swap of DNA segments. Instead, one sequence is corrected or replaced while the related partner sequence remains...

restarting stalled replication forks

Restarting DNA Replication After Stalls

DNA replication can stop before it is finished when the fork stalls. A stalled replication fork is a point where the DNA-copying machinery slows down or pauses during chromosome duplication. Cells can restart these forks to keep copying DNA. This process helps the replication machinery move forward again after the pause. It is an important part of maintaining normal DNA replication when problems arise during copying. The term stalled replication fork describes the stopped or delayed state of...

key terms and concepts

Learn how DNA polymerase proofreads new DNA, removes mismatched bases, and how reduced proofreading can raise mutation rates or be used in PCR.

DNA Polymerase Error Correction | JoVE K12

cell biology

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Pennsylvania State Academic Standards

Life Science

Inheritance and Variation of Traits

3.1.9-12.Q

Louisiana Student Standards for Science

High School

Life Sciences

Heredity: Inheritance And Variation Of Traits

HS-LS3-3

Alabama Course of Study Science

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

From Molecules to Organisms: Structures and Processes

9-12.B.1.3.a

Florida Department of Education

SC.912.L.16

SC.912.L.16.3

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Use models to explain how DNA is passed from parents to offspring through the processes of meiosis and fertilization in sexual reproduction.

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genome-size-and-the-evolution-of-new-genes

Genome Size and the Evolution of New Genes

types-of-genetic-transfer-between-organisms

Types of Genetic Transfer Between Organisms

Base-pairing and DNA Repair

The DNA Replication Fork

Lagging Strand Synthesis

The Replisome

Replication in Prokaryotes

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the ...

Replication in Eukaryotes

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Overview of DNA Repair

Base Excision Repair

Nucleotide Excision Repair

DNA Distortion and Damage
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Mismatch Repair

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Fixing Double-strand Breaks

Homologous Recombination

Gene Conversion

Restarting Stalled Replication Forks

Construct an explanation to summarize how inheritable genetic variations may result from: new genetic combinations in meiosis, mutations during replication, or mutations caused by environmental factors.

An Overview of Genetic Analysis

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Genetic Crosses

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Punnett Squares

Monohybrid Crosses

Dihybrid Crosses

Trihybrid Crosses

Trihybrid Crosses
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Law of Independent Assortment

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Pedigree Analysis

Multiple Allele Traits

Incomplete Dominance

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Lethal Alleles

Agouti: A Lethal Allele
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Polygenic Traits

background-and-environment-affect-phenotype

Background and Environment Affect Phenotype

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Use mathematics and computational thinking to predict the variation and distribution of expressed traits based on: Mendelian inheritance, co-dominance, incomplete dominance, multiple alleles, and sex-linked inheritance.

LS.Bio.7.1

Overview

A pedigree is a diagram displaying a family&#x2019;s history of a trait. Analyzing pedigrees can reveal (1) whether a trait is dominant or ...

Probability Laws

Overview

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The Concept of Multiple Allelism

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Analyze and interpret data to explain how polygenic traits result in a wide range of phenotypes.

LS.Bio.7.2

Pleiotropy

Pleiotropy is the phenomenon in which a single gene impacts multiple, seemingly unrelated phenotypic traits. For example, defects in the SOX10 gene cause ...

Nature and Nurture

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LS.Bio.7.3

PCR: The Polymerase Chain Reaction

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DNA Gel Electrophoresis

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Restriction Enzymes

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DNA Agarose Gel Electrophoresis

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dna-isolation-and-restriction-enzyme-analysis

DNA Isolation and Restriction Enzyme Analysis

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Preparation of Solutions and Materials
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DNA Isolation
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Analyze and interpret data to compare DNA samples.

LS.Bio.8.1

Transgenic Organisms

Reproductive Cloning

CRISPR

Synthetic Biology

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Obtain and communicate information that summarizes the impact of biotechnology applications on the individual, society, and the environment, including agriculture and medicine.

LS.Bio.8.2

Understand genetic mechanisms for variation.

DNA Polymerase Error Correction

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