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DNA Repair in Replication*# - Biology

DNA Repair in Replication*# - Biology


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Replication design challenge: proofreading

When the cell begins the task of replicating the DNA, it does so in response to environmental signals that tell the cell it is time to divide. This is a daunting task when you consider that there are ~6,500,000,000 base pairs in the human genome and ~4,500,000 base pairs in the genome of a typical E. coli strain and that Nature has determined that the cells must replicate within 24 hours and 20 minutes, respectively. In either case, many individual biochemical reactions need to take place.

While ideally replication would happen with perfect fidelity, DNA replication, like all other biochemical processes, is imperfect—bases may be left out, extra bases may be added, or bases may be added that do not properly base-pair. In many organisms, many of the mistakes that occur during DNA replication are promptly corrected by DNA polymerase itself via a mechanism known as proofreading. In proofreading, the DNA polymerase "reads" each newly added base via sensing the presence or absence of small structural anomalies before adding the next base to the growing strand. In doing so, a correction can be made.

If the polymerase detects that a newly added base has paired correctly with the base in the template strand, the next nucleotide is added. If, however, a wrong nucleotide is added to the growing polymer, the misshaped double helix will cause the DNA polymerase to stall, and the newly made strand will be ejected from the polymerizing site on the polymerase and will enter into an exonuclease site. In this site, DNA polymerase is able to cleave off the last several nucleotides that were added to the polymer. Once the incorrect nucleotides have been removed, new ones will be added again. This proofreading capability comes with some trade-offs: using an error-correcting/more accurate polymerase requires time (the trade-off is speed of replication) and energy (always an important cost to consider). The slower you go, the more accurate you can be. Going too slow, however, may keep you from replicating as fast as your competition, so figuring out the balance is key.

Errors that are not corrected by proofreading become what are known as mutations.

Figure 1. Proofreading by DNA polymerase corrects errors during replication.

Suggested discussion

Why would DNA replication need to be fast? Consider the environment the DNA is in, and compare that to the structure of DNA while being replicated.

Suggested discussion

What are the pros and cons of DNA polymerase's proofreading capabilities?

Replication mistakes and DNA repair

Although DNA replication is typically a highly accurate process, and proofreading DNA polymerases helps to keep the error rate low, mistakes still occur. In addition to errors of replication, environmental damage may also occur to the DNA. Such uncorrected errors of replication or environmental DNA damage may lead to serious consequences. Therefore, Nature has evolved several mechanisms for repairing damaged or incorrectly synthesized DNA.

Mismatch repair

Some errors are not corrected during replication but are instead corrected after replication is completed; this type of repair is known as a mismatch repair. Specific enzymes recognize the incorrectly added nucleotide and excise it, replacing it with the correct base. But, how do mismatch repair enzymes recognize which of the two bases is the incorrect one?

In E. coli, after replication, the nitrogenous base adenine acquires a methyl group; this means that directly after replication the parental DNA strand will have methyl groups, whereas the newly synthesized strand lacks them. Thus, mismatch repair enzymes are able to scan the DNA and remove the wrongly incorporated bases from the newly synthesized, non-methylated strand by using the methylated strand as the "correct" template from which to incorporate a new nucleotide. In eukaryotes, the mechanism is not as well understood, but it is believed to involve recognition of unsealed nicks in the new strand, as well as a short-term, continuing association of some of the replication proteins with the new daughter strand after replication has completed.

Figure 2. In mismatch repair, the incorrectly added base is detected after replication. The mismatch repair proteins detect this base and remove it from the newly synthesized strand by nuclease action. The gap is now filled with the correctly paired base.

Nucleotide excision repair

Nucleotide excision repair enzymes replace incorrect bases by making a cut on both the 3' and 5' ends of the incorrect base. The entire segment of DNA is removed and replaced with correctly paired nucleotides by the action of a DNA polymerase. Once the bases are filled in, the remaining gap is sealed with a phosphodiester linkage catalyzed by the enzyme DNA ligase. This repair mechanism is often employed when UV exposure causes the formation of pyrimidine dimers.

Figure 3. Nucleotide excision repairs thymine dimers. When exposed to UV, thymines lying adjacent to each other can form thymine dimers. In normal cells, they are excised and replaced.

Consequences of errors in replication, transcription, and translation

Something key to think about:

Cells have evolved a variety of ways to make sure DNA errors are both detected and corrected. We have already discussed several of them. But why did so many different mechanisms evolve? From proofreading by the various DNA-dependent DNA polymerases, to the complex repair systems. Such mechanisms did not evolve for errors in transcription or translation. If you are familiar with the processes of transcription and/or translation, think about what the consequences would be of an error in transcription. Would such an error affect the offspring? Would it be lethal to the cell? What about errors in translation? Ask the same questions about the process of translation. What would happen if the wrong amino acid is accidentally put into the growing polypeptide during translation? How do these contrast with DNA replication? If you are not familiar with transcription or translation, don't fret. We'll learn those soon and return to this question again.


DNA repair and genome maintenance in Bacillus subtilis

From microbes to multicellular eukaryotic organisms, all cells contain pathways responsible for genome maintenance. DNA replication allows for the faithful duplication of the genome, whereas DNA repair pathways preserve DNA integrity in response to damage originating from endogenous and exogenous sources. The basic pathways important for DNA replication and repair are often conserved throughout biology. In bacteria, high-fidelity repair is balanced with low-fidelity repair and mutagenesis. Such a balance is important for maintaining viability while providing an opportunity for the advantageous selection of mutations when faced with a changing environment. Over the last decade, studies of DNA repair pathways in bacteria have demonstrated considerable differences between Gram-positive and Gram-negative organisms. Here we review and discuss the DNA repair, genome maintenance, and DNA damage checkpoint pathways of the Gram-positive bacterium Bacillus subtilis. We present their molecular mechanisms and compare the functions and regulation of several pathways with known information on other organisms. We also discuss DNA repair during different growth phases and the developmental program of sporulation. In summary, we present a review of the function, regulation, and molecular mechanisms of DNA repair and mutagenesis in Gram-positive bacteria, with a strong emphasis on B. subtilis.

Figures

Model for activation of the…

Model for activation of the SOS response in B. subtilis. (A) In this…

Model for repair of a single double-strand break by homologous recombination in B.…

Schematic representation of the domain…

Schematic representation of the domain structure of B. subtilis DNA helicases RecQ and…

Model for double Holliday junction…

Model for double Holliday junction formation during homologous recombination and repair of DSBs…

Crystal structure of Holliday junction…

Crystal structure of Holliday junction resolvase RecU of B. subtilis. (Adapted from reference…

Model for mismatch repair in…

Model for mismatch repair in B. subtilis. (A and B) The β clamp…

Schematic diagram of the genome…

Schematic diagram of the genome maintenance checkpoints in B. subtilis. (A) The interplay…


DNA Replication is Semiconservative and Initiates at Origins of Replication

F igure 1. Semiconservative model of DNA replication. DNA replication is semiconservative. Each strand of a double-stranded molecule is used as a template for DNA replication. Therefore, each newly replicated DNA is composed of one strand of newly synthesized DNA and one strand of the conserved DNA from the original molecule.

Figure 2. DNA synthesis. DNA synthesis requires a template strand and it always occurs in the 5' to 3' direction. The incoming deoxyribonucleotide is added to the 3' hydroxyl group of the last nucleotide in the strand.

The structure of double-stranded DNA supports a model for DNA replication (see Tutorial entitled DNA and Chromosomes), in which each strand of the DNA is used as a template (is copied to make the complementary strand). This is, in fact, the mechanism of DNA replication, and it is referred to as semiconservative replicationbecause each new double-stranded DNA molecule is composed of one strand of the conserved "old" DNA and one strand of newly synthesized DNA (seeFigure 1). The template DNA is used as a blueprint for the sequence of the new strand, following the rules of complementary bases (e.g. if the nucleotide is adenine in the template DNA, then a thymine is added in the opposite strand). DNA polymerase (the enzyme that carries out DNA synthesis) uses one strand of DNA as a template and adds a new nucleotide to the 3' end of the new elongating strand. DNA polymerase utilizes a deoxyribonucleotide, cleaves the two terminal phosphates from the 5' end of the nucleotide, and uses the free energy to form a phosphodiester bond between the 5' phosphate of the incoming nucleotide and the 3' hydroxyl end of the last nucleotide in the strand (Figure 2). Therefore, the newly synthesized DNA strand can only elongate in one direction, 5' to 3'.

Figure 3. Origins of replication. Replication is initiated at a specific site in the chromosome called the origin of replication (ORI). The replication fork is the site of the progressive unwinding of the double-stranded DNA and the duplication of the template DNA, and it travels along the length of the DNA molecule, away from the ORI. In a circular DNA molecule (bacterial DNA), there is a single ORI that spreads bidirectionally throughout the molecule. In a linear DNA molecule (a chromosome), there are multiple ORI that spread bidirectionally along the chromosome, away from the ORI, until they reach another replication fork moving in the opposite direction. DNA synthesis is distinct for each of the template strands the leading strand directs continuous synthesis of the new strand, whereas the lagging strand directs discontinuous DNA synthesis. The fragments of DNA generated from the lagging strand are referred to as Okazaki fragments.

Recall, in chromosomes, DNA replication is initiated at origins of replication (ORI) scattered along the length of the chromosomes (Figure 3). At the ORI, the DNA is locally denatured and a complex of proteins is assembled on the DNA to carry out DNA synthesis. Although the DNA sequence of any individual ORI may vary, there are some common features: the ORI sequence is composed of multiple, short, repeated sequences proteins bind to these sequences and recruit DNA polymerase and the other proteins necessary for DNA replication and the stretches of DNA that flank the ORI have a high percentage of the nucleotides adenine and thymine, which facilitate the unwinding of the DNA molecule. Once assembled at the ORI, the DNA replication protein complex will travel along the DNA, unwinding and copying it in what is termed the replication fork (see Figure 3). Replication forks move from the ORI in both directions (to the right and left of the ORI) in a fashion referred to as bidirectional replication. At each replication fork, the double-stranded DNA is unwound and each strand of DNA serves as a template. If one examines the overall direction of replication (5' to 3') and the polarity of the two template strands (see Figure 3), it becomes clear that the replication fork is asymmetric. Remember that DNA polymerase can only elongate DNA in the 5' to 3' direction (that is, read the template in the 3' to 5' direction), so one template strand can be read (and direct continuous synthesis of its complementary strand) in the same direction as the overall direction of replication. The template strand used for the continuous synthesis of its complementary strand is termed the leading strand. The other strand is termed the lagging strand, and the synthesis of its complementary strand occurs in the opposite direction of the overall direction of replication. The lagging strand template directs the discontinuous elongation of its complementary DNA, resulting in short fragments of complementary DNA termed Okazaki fragments. The Okazaki fragments are eventually linked together to generate a single, continuous strand of DNA (described later in this tutorial).


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Your Infringement Notice may be forwarded to the party that made the content available or to third parties such as ChillingEffects.org.

Please be advised that you will be liable for damages (including costs and attorneys’ fees) if you materially misrepresent that a product or activity is infringing your copyrights. Thus, if you are not sure content located on or linked-to by the Website infringes your copyright, you should consider first contacting an attorney.

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Biologists unravel full sequence of DNA repair mechanism

Credit: Photo by Daniil Kuzelev/Unsplash.

Every living organism has DNA, and every living organism engages in DNA replication, the process by which DNA makes an exact copy of itself during cell division. While it's a tried-and-true process, problems can arise.

Break-induced replication (BIR) is a way to solve those problems. In humans, it is employed chiefly to repair breaks in DNA that cannot be fixed otherwise. Yet BIR itself, through its repairs to DNA and how it conducts those repairs, can introduce or cause genomic rearrangements and mutations contributing to cancer development.

"It's kind of a double-edged sword," says Anna Malkova, professor in the Department of Biology at the University of Iowa, who has studied BIR since 1995. "The basic ability to repair is a good thing, and some DNA breaks can't be repaired by other methods. So, the idea is very good. But the outcomes can be bad."

A new study led by Malkova, published Jan. 20 in the journal Nature, seeks to tease out BIR's high risk-reward arrangement by describing for the first time the beginning-to-end sequence in BIR. The biologists developed a new technique that enabled them to study in a yeast model how BIR operates throughout its repair cycle. Until now, scientists had only been able to study BIR's operations at the beginning and end stages. The researchers then introduced obstructions with DNA replication, such as transcription—the process of copying DNA to produce proteins—that are believed to be aided by BIR.

"Our study shows that when BIR comes to the rescue at these collisions, its arrival comes at a very high price," says Malkova, the study's corresponding author. "When BIR meets transcription, it can introduce even more instability, which can lead to even higher mutations. As a result, we think that instabilities that mainly were found at collisions between transcription and replication that have been suggested to lead to cancer might be caused by BIR that came to the rescue. It comes, it rescues, but it's kind of questionable how helpful it really is."

Scientists have known how BIR works at some stages. For example, they know the DNA repair apparatus forms a bubble of sorts around the damaged DNA, then moves forward, unzipping the DNA, copying intact segments, and finally transferring those copied segments to a new DNA strand.

But what remained elusive was following BIR throughout its entire repair cycle. Using a technique involving Droplet Digital PCR and a new DNA purification method developed by biology graduate student Liping Liu, the researchers were able to observe BIR from beginning to end.

"If you imagine this as a train, Liping installed a bunch of stations, and she watched how the train proceeded at each station, tracking the increase in DNA at each station, how much increase is occurring at each station, and thus, in aggregate, how the entire process unfolds," Malkova explains.

The team then intentionally introduced obstructions at some stations—transcription and another obstruction called internal telomere sequences—to observe how BIR responded to the obstacles. One finding: when transcription is introduced near the beginning of the BIR process, the repairs fail to commence, as if they're being suppressed. Also, the researchers found the orientation of the transcription with respect to BIR can affect the repair cycle and may be an important factor affecting instability that can promote cancer in humans.

"Scientists already know there's a lot of instability in places where high transcription meets normal replication," Malkova says. "What we did not know until now is where is it coming from and why is it happening."

The first author of the study, "Tracking break-induced replication shows that it stalls at roadblocks," is Liu, who is a sixth-year graduate student in Malkova's lab.


Molecular Biology 4 - DNA replication, Repair, recombination

the major initiator protein, the helices, the primase.

in the first step several molecules of the initiator protein bind to specific DNA sequences at the replication origin and destabilize the double helix by forming a compact structure in which the DNA is tightly wrapped around the protein..

Next two helices are brought in by the helices loading proteins, which inhibit the helices until they are properly loaded at the replication origin.

Helices loading proteins prevent the replicative DNA helices rom inappropriately entering other single strand stretches of DNA in the bacterial genome.

Aided by SSBP the loaded helices open up the DNA thereby enabling promises to enter and synthesize initial primers.

In subsequent steps two complete replication forks are assembled at the origin and move off in opposite direction.


Abstract

Post-translational modification by ubiquitin is best known for its role in targeting its substrates for regulated degradation. However, non-proteolytic functions of the ubiquitin system, often involving either monoubiquitylation or polyubiquitylation through Lys63-linked chains, have emerged in various cell signalling pathways. These two forms of the ubiquitin signal contribute to three different pathways related to the maintenance of genome integrity that are responsible for the processing of DNA double-strand breaks, the repair of interstrand cross links and the bypass of lesions during DNA replication.


VISUAL CONNECTION

Figure 3: A replication fork is formed by the opening of the origin of replication, and helicase separates the DNA strands. An RNA primer is synthesized, and is elongated by the DNA polymerase. On the leading strand, DNA is synthesized continuously, whereas on the lagging strand, DNA is synthesized in short stretches. The DNA fragments are joined by DNA ligase (not shown).

You isolate a cell strain in which the joining together of Okazaki fragments is impaired and suspect that a mutation has occurred in an enzyme found at the replication fork. Which enzyme is most likely to be mutated? The answer is ligase, as this enzyme joins together Okazaki fragments.

Telomere Replication

Because eukaryotic chromosomes are linear, DNA replication comes to the end of a line in eukaryotic chromosomes. As you have learned, the DNA polymerase enzyme can add nucleotides in only one direction. In the leading strand, synthesis continues until the end of the chromosome is reached however, on the lagging strand there is no place for a primer to be made for the DNA fragment to be copied at the end of the chromosome. This presents a problem for the cell because the ends remain unpaired, and over time these ends get progressively shorter as cells continue to divide. The ends of the linear chromosomes are known as telomeres , which have repetitive sequences that do not code for a particular gene. As a consequence, it is telomeres that are shortened with each round of DNA replication instead of genes. For example, in humans, a six base-pair sequence, TTAGGG, is repeated 100 to 1000 times. The discovery of the enzyme telomerase (Figure 4) helped in the understanding of how chromosome ends are maintained. The telomerase attaches to the end of the chromosome, and complementary bases to the RNA template are added on the end of the DNA strand. Once the lagging strand template is sufficiently elongated, DNA polymerase can now add nucleotides that are complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.

Figure 4: The ends of linear chromosomes are maintained by the action of the telomerase enzyme.

Telomerase is typically found to be active in germ cells, adult stem cells, and some cancer cells. For her discovery of telomerase and its action, Elizabeth Blackburn (Figure 5) received the Nobel Prize for Medicine and Physiology in 2009.

Figure 5: Elizabeth Blackburn, 2009 Nobel Laureate, was the scientist who discovered how telomerase works. (credit: U.S. Embassy, Stockholm, Sweden)

Telomerase is not active in adult somatic cells. Adult somatic cells that undergo cell division continue to have their telomeres shortened. This essentially means that telomere shortening is associated with aging. In 2010, scientists found that telomerase can reverse some age-related conditions in mice, and this may have potential in regenerative medicine. 1 Telomerase-deficient mice were used in these studies these mice have tissue atrophy, stem-cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved functioning of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.

DNA Replication in Prokaryotes

Recall that the prokaryotic chromosome is a circular molecule with a less extensive coiling structure than eukaryotic chromosomes. The eukaryotic chromosome is linear and highly coiled around proteins. While there are many similarities in the DNA replication process, these structural differences necessitate some differences in the DNA replication process in these two life forms.

DNA replication has been extremely well-studied in prokaryotes, primarily because of the small size of the genome and large number of variants available. Escherichia coli has 4.6 million base pairs in a single circular chromosome, and all of it gets replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the chromosome in both directions. This means that approximately 1000 nucleotides are added per second. The process is much more rapid than in eukaryotes. Table 1 summarizes the differences between prokaryotic and eukaryotic replications.

Table 1: Differences between Prokaryotic and Eukaryotic Replications
Property Prokaryotes Eukaryotes
Origin of replication Single Multiple
Rate of replication 1000 nucleotides/s 50 to 100 nucleotides/s
Chromosome structure circular linear
Telomerase Not present Present


Enzymes and Proteins Involved in the DNA Replication

A number of enzymes and proteins are associated with the replication fork to help in the initiation and continuation of DNA synthesis, Most prominently, DNA polymerase synthesizes the new strands by adding nucleotides that complement each (template) strand.
DNA replication occurs during the S-phase of interphase. At the replication fork, many replication enzymes assemble on the DNA into a complex molecular machine called the replisome. The following is a list of major DNA replication enzymes that participate in the replisome.

1) DNA Helicase :

2) Single Stranded Binding Proteins (SSB proteins) :

3). Topoisomerase :

4).DNA Gyrase :

5). Primase :

6) DNA Polymerase :

A). Prokaryotic DNA polymerase -

DNA polymerase I -
It is made up of one subunits. It has 3' to 5' and 5' to 3' exonuclease activity.
Function - DNA repair, Gap filling and synthesis of new lagging strand.

DNA polymerase II -
It is made up of 7 subunits. It has only 3' to 5' exonuclease activity.
Function - DNA repair and DNA proof reading.

DNA polymerase III -
It is made up of at least 10 subunits. It has 3' to 5' exonuclease activity.
Function - This is the main replication enzyme in Prokaryotes.

b) Eukaryotic DNA polymerase -
Eukaryotes has 5 types of DNA polymerase which are DNA polymerase α, β, γ, δ and ε.

DNA polymerase α -
It has no any exonuclease activity.
Function - DNA replication in the nucleous.

DNA polymerase β -
It has no any exonuclease activity.
Function - DNA replication and base excision repair.

DNA polymerase γ -
It has 3' to 5' exonuclease activity.
Function - DNA replication in Mitochondria.

DNA polymerase δ -
It has 3' to 5' exonuclease activity.
Function - Synthesis of lagging strand during DNA replication.

DNA polymerase ε -
It has 3' to 5' exonuclease activity.
Function - Synthesis of leading strand during DNA replication.


Watch the video: Repairing Damaged DNA by Recombination (May 2022).