Information

How do catenanes form when DNA replicates?


So I am taking a course in DNA replication and repair. And we are talking about catenanes forming when DNA replicates (two circles of dsDNA interlinked) How is this possible?


The first DNA circle is double-stranded. If you could melt the double-helix completely you would not be able to pull the two stands apart without breaking the sugar-phosphate backbone of at least one of the two strands. This is a topological problem, the two strands are linked to each other.

Now consider DNA replication. In the simplest example there is a single origin of replication and there will be two replication forks proceeding around the circular genome in opposite directions. This is exactly what happens during DNA replication in the bacterium E. coli. The DNA double helix of the parental molecule "melts", the RNA primers are synthesized by primase, and the DNA polymerase complexes initiate synthesis. Behind each replication fork there are now two hybrid DNA strands, each with a parental strand, and a new daughter strand.

Thus far no sugar-phosphate bonds have been cleaved. When replication is done each daughter ds DNA helix will have a single-stranded nick at the origin, and another one at the site where replication terminated, but those will be quickly repaired by DNA polymerase I and DNA ligase. Even if those two repair enzymes were inhibited, and you could somehow melt the DNA strands of both the daughter chromosomes, you would only be able to pull out the new DNA molecules (with the nicks), the two original parental strands are still topologically linked.

To resolve two concatenated DNA circles you need to make a double-stranded break in at least one of the ds circles. This enzymatic activity is provided by a class of enzymes named Type II DNA topoisomerases.


Topological challenges to DNA replication: Conformations at the fork

The unwinding of the parental DNA duplex during replication causes a positive linking number difference, or superhelical strain, to build up around the elongating replication fork. The branching at the fork and this strain bring about different conformations from that of (−) supercoiled DNA that is not being replicated. The replicating DNA can form (+) precatenanes, in which the daughter DNAs are intertwined, and (+) supercoils. Topoisomerases have the essential role of relieving the superhelical strain by removing these structures. Stalled replication forks of molecules with a (+) superhelical strain have the additional option of regressing, forming a four-way junction at the replication fork. This four-way junction can be acted on by recombination enzymes to restart replication. Replication and chromosome folding are made easier by topological domain barriers, which sequester the substrates for topoisomerases into defined and concentrated regions. Domain barriers also allow replicated DNA to be (−) supercoiled. We discuss the importance of replicating DNA conformations and the roles of topoisomerases, focusing on recent work from our laboratory.

A thorough understanding of DNA replication and recombination requires knowledge of the conformations and topology of replicating DNA. These are different from those of nonreplicating DNA. The action of DNA helicases, interruptions in replicated strands, and, most importantly, the uniquely branched structure of the replication fork itself, all contribute to these differences. In this review, we illustrate the major conformational differences between replicating and nonreplicating DNA and their physiological importance. We highlight the evidence for each structure in vitro and in vivo. In addition, we address how the links originally residing in the double helix of the parental duplex are fully resolved in bacteria by two type-2 topoisomerases, DNA gyrase and topoisomerase (topo) IV, to form two separate daughter molecules. Although we emphasize the situation in bacteria, we will also make generalizations applicable to the eukarya and archaea.

We begin by defining a few basic terms that form the language of DNA topology (1). The topology we will focus on are the links between the complementary Watson and Crick strands of an intact, topologically constrained piece of DNA. The simplest example is a closed circular DNA, as is found in plasmids and viruses, but the results can be generalized to linear chromosomes because of their organization into closed domains or loops. The intertwining of the complementary strands is described by the linking number (Lk), which is one-half of the signed number of times one strand crosses the other in any projection. According to the sign convention, the crossings in ordinary B-type DNA are (+). The crossings, or nodes, of the complementary strands can result from the local intertwining of the double helix itself, in which case they are measured by a parameter called twist (Tw). Alternatively, nodes result from one segment of the double helix crossing another, as measured by writhe (Wr). Lk is the sum of Tw and Wr. Notably, Lk is unaltered by any deformation short of DNA breakage and reunion. More important is the quantity ΔLk, the difference between Lk and Lk0, where Lk0 is the Lk of a relaxed DNA molecule. The strain on the DNA from a non-zero ΔLk often causes the DNA to supercoil, a form of writhe. Supercoiling can be either plectonemic (interwound) or solenoidal, as when DNA wraps around a protein. The most useful measure of the topological deviation of DNA from the relaxed state is its supercoiling density, or σ. Sigma is equal to ΔLk/Lk0 and is therefore independent of DNA length. Replication causes an increase in ΔLk, because separation of the parental strands lowers the value of Lk0. Therefore, the ΔLk of replication increases by about one for every ten base pairs of replicated DNA.

This review is divided into three parts. We begin by discussing the conformations of (−) supercoiled DNA that is not replicating, the form that DNA adopts away from the fork. Next, we discuss the three ways in which replicating DNA may differ from nonreplicating DNA: precatenanes, (+) supercoiled DNA, and the four-way junction at stalled forks. Finally, we will discuss topological domain barriers, which can sequester replicating DNA structures into limited regions of the chromosome where they can be processed more readily, allowing replication and chromosome segregation to proceed.


DNA Replication: Eukaryotic Elongation and Termination.

We discussed about the Initiation in the Eukaryotic cells in the last post. In the present post, let’s look into the Elongation and the Termination in Eukaryotic DNA Replication.

During Initiation, a repertoire of proteins bind and unwind the DNA at the origin. To this protein complex the polymerases are loaded. The polymerases work together with other proteins for the elongation of the daughter strands.

(Just for info: Read more about the eukaryotic origins in the paper titled ‘Making Sense of Eukaryotic DNA Replication Origins‘.)

Elongation:

The first polymerase to initiate the DNA synthesis is the DNA polymerase α, which exists in the form of DNA polymerase α-primase complex. The primase subunit synthesizes the RNA primers (around 7-12 nucleotides) which are then transferred to the polymerase domain and extended with DNA bases (around 20-25 nucleotides). Replication factor C (RFC) initiates a reaction called polymerase switching. The DNA strands are then extended by other polymerases namely the DNA polymerase δ and DNA polymerase ε.

Leading strand synthesis:

Fig 1: Synthesis of leading or continuous strand.

As DNA pol α completes synthesizing RNA primer and adding DNA bases, the RFC causes dissociation of DNA pol α and assembles proliferating cell nuclear antigen (PCNA) in the region of the primer terminus. PCNA is a DNA clamp for DNA polymerases.

Pol ε has been reported as the main leading strand synthesis polymerase (in Saccharomyces cerevisiae). In the leading strand as the replication is continuous and the primer is synthesized only once and the extension is carried out (fig 1).

Lagging strand synthesis:

Fig 2: Synthesis of lagging or discontinuous strand with series of Okazaki fragments.

Polymerases δ is the major polymerase in lagging-strand synthesis. The replication in the lagging strands is discontinuous and takes place with formation of several Okazaki fragments (fig 2).

Okazaki fragments (fig 3) generated in eukaryotes during lagging-strand synthesis are around 200 bases (prokaryotes, around 2000 bases) long. As several Okazaki fragments are made, Polymerase switching during synthesis of Okazaki fragments is of higher importance.

Hence an Okazaki fragment is made up of RNA nucleotides (7-10 nucleotides), then around 10-20 nucleotides of DNA bases are added by the DNA pol α. Following which, the RFC causes polymerase switching and the deoxyribonucleotides are added by DNA pol δ held by the PCNA, the sliding clamp (see the figure below). DNA pol δ dissociates after the synthesis of the entire DNA stretch, as it approaches the previous RNA primer.

(Just for info: Know more about PCNA)

The proteins involved in the replication, especially PCNA (Boehm et al., 2016).

RNA primers are removed by RNase H1, such that one ribonucleotide remains still attached to the DNA (3′ end) part of the Okazaki fragment. This left out ribonucleotide is then removed by flap endonuclease 1 (FEN 1). Polymerase δ then fills the gaps formed between Okazaki fragments following the primer removal. The nick formed between the Okazaki fragment and the lagging strand are filled in by DNA ligase I, hence forming single lagging strand.

Fig 4: Removal of RNA primer and linking of individual Okazaki fragments.

Nucleosome Assembly:

During elongation, there is continuous disassembly as reassembly of the nucleosome packaging along the DNA, too. As we know, one of the feature of the eukaryotic DNA is that it is packaged in form of highly compact structures called the Chromosomes. It involves winding of the negatively charged DNA around the basic proteins called histones to form structures called nucleosomes (fig below). Each nucleosome is composed of 8 histone proteins, two each of H2A, H2B, H3 and H4. Histone H1 forms the linker between two nucleosomes.

The nucleosomes.

During replication, two nucleosomes in front of the replication fork, that is unreplicated DNA, becomes destabilized. Hence the replication fork movement causes DNA packaging to disorganise to allow the replication proteins to interact with the DNA.

In the replicated portion, the leading and lagging strands were nucleosome-free till about 225 bp and 285 bp, respectively. Following this region, the DNA was packaged with histone octomer but some lacked histone H1. After around 450 bp to 650bp from the replication fork, complete nucleosomes with H1 were detected in the daughter strands. Hence there is stepwise assembly of daughter strands into complete nucleosome.

The parental nucleosomes disassociate and randomly bind the daughter DNA strands, such that each strands has half of the parental nucleosomes. The remaining nucleosome components required for packaging the two daughter strands, are synthesized de novo and assembled onto the daughter strands.

Termination:

Following the successful elongation, the replication has to be terminated to give two separate copies of the DNA.

Involving two adjacent replication forks:

As the eukaryotic cell have a large number of origins, the termination involves merging of two adjacent replication forks. This includes four different steps:

– Dissolution:

The DNA stretch between the two adjacent forks (fig 5.a) is unwound (fig 5.b) and the approaching CMGs pass each other (fig 5.c). The last Okazaki fragment is processed by DNA pol δ and FEN1 (fig 5.d).

The gaps in the daughter strands (as in normal Okazaki fragments) are filled in and two oppositely approaching synthesized strands are ligated.

– Dissociation of replisome:

The replisome complex dismantles after the convergence of the two replication fork. This process involves termination-specific polyubiquitylation of Mcm7 and the p97/VCP/Cdc48 segregase (fig 5.e).

– Decatenation:

If there are any intertwinings in the daughter DNA strands or catenanes, they are removed utilising topoisomerase II segregating the two strands.

Fig 5: Termination involves merging of the two neighbouring forks (Dewar & Walter, 2017).

Involving the ends of the Chromosomes:

As is known the DNA in eukaryotic chromosomes is a linear molecule, the termination in eukaryotic DNA also involve completing replication at the ends of chromosomes known as Telomeres (fig 6).

Fig 6: Telomeres form protective end of eukaryotic linear DNA (Aulinas, 2013).

During the synthesis of Okazaki fragments, RNA primer provide 3′-OH group for 5′ to 3′ replication. On the removal of RNA primer, from the lagging strand at the chromosome end, the end remains unreplicated and the newly synthesized strand is shortened (fig 7).

Fig 7: Shortening of the chromosome ends.

This shortening of the chromosome is prevented by the presence of special repeats of sequences called telomeres (and telomere-associated proteins) at the ends of DNA in chromosomes contain. For e.g. human Chromosomes are protected by telomeres having repeated sequences of (TTAGGG)n of about 15–20 kb at birth. These structures protect the ends of chromosomes from being mistakenly considered as DNA double strand breaks (DSB).

In normal somatic cells, the telomeric region of eukaryotic chromosomes are shortened with each round of DNA replication. After certain number of DNA replications, and hence cell divisions, the telomeres are shortened to an extent that it leads to replicative cell senescence or apoptosis.

(Just for info: Please read about the Nobel prize given to a work on telomeres.)

However, in germline and cancer cells, an enzyme called telomerase, extends the ends of the chromosomes, especially the 5′ end of the lagging strands. The ability to maintain the length of the telomeres, make these cells immortal.

Telomerase is a reverse transcriptase which has a RNA template, known as template-encoding RNA molecule (TER) for extension of the telomeric DNA (fig 8). The basic protein component of telomerase is known as TERT (TElomerase Reverse Transcriptase).

In humans, the RNA template has the sequence AUCCCAAUC. The newly synthesized telomeric DNA repeats are added to the overhanging single-stranded 3′ end of the DNA (fig 8).

Fig 8: Synthesis of telomeric DNA repeats by Telomerase (Verhoeven et al, 2014).

(Just for info: visit to watch the animation on action of Telomerase and much more.)

After strand extension on the 3′ end by the telomerase is completed, DNA pol α and DNA ligase complete the DNA strand synthesis.

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Read more posts by The Biotech Notes:

Bambara et al. (1997) Enzymes and Reactions at the Eukaryotic DNA Replication Fork. J Biol Chem. 272(8):4647-50.

Abmayr and Workman (2012) Holding on through DNA Replication: Histone Modification or Modifier? Cell. 150(5):875-7.

Verhoeven et al (2014). Cellular aging in depression: Permanent imprint or reversible process?: An overview of the current evidence, mechanistic pathways, and targets for interventions. BioEssays 36(10):968-78.

Bailey et al (2015) Termination of DNA replication forks: “Breaking up is hard to do”.Nucleus 6 (3):187-196.

Dewar & Walter (2017) Mechanisms of DNA replication termination. Nature Reviews Molecular Cell Biology 18:507–516.

Aulinas (2013) Telomeres, aging and Cushing’s syndrome: Are they related? Endocrinology and Nutrition (English Edition) 60(6): 329-335.

Boehm et al. (2016) The Many Roles of PCNA in Eukaryotic DNA Replication. Enzymes 39:231-54.


DNA synthesis

The mechanism of DNA replication is greatly influenced by DNA structure. The complementary base pairing between the nitrogen bases A-T and G-C underlies the semi-conservative nature of DNA replication, which results in a duplicated genome with one parental strand and one newly synthesized strand. Each strand serves as a template for the DNA polymerase to catalyze the addition of the correct base during synthesis of a new complementary strand. As the strands are antiparallel with opposing polarity and since DNA polymerases can only synthesize DNA in the 5&prime to 3&prime direction, only one strand is continuously synthesized. This strand is called the leading strand. Synthesis of the other strand, called the lagging strand, is made possible through discontinuous synthesis of short fragments, called Okazaki fragments, in the 5&prime to 3&prime direction, which are later joined together.

The replicating DNA: DNA replication proteins at the replication fork. The helicase unwinds the duplex DNA and Single Strand Binding proteins (SSBs) coat and stabilize single stranded DNA formed by strand separation. Topoisomerase is seen ahead of the fork removing superhelical tension caused by strand separation. Note that the leading strand is synthesized continuously in the 5&prime to 3&prime direction, whereas the lagging strand is synthesized discontinuously as short fragments called Okazaki fragments. The Polymerase &alpha-primase complex synthesizes short RNA primers that are extended up to 30-40 nucleotides. Thereafter polymerase &epsilon and polymerase &delta takes up the job of faster and efficient strand synthesis on lagging and leading strands respectively. Ligase seals the gap between Okazaki fragments.

DNA synthesis begins in S phase as the replicative helicase unwinds and separates the two strands of the DNA double helix [7] . As the helicase unwinds DNA, DNA polymerase synthesizes DNA utilizing the exposed single stranded DNA as a template. DNA polymerases &lsquoread&rsquo the template strand and add the correct complimentary base. Energy for polymerization comes from release of a pyrophosphate from a free deoxyribonucleotide triphosphate (dNTP), creating a 5&primemonophosphate that could be covalently linked to the 3&prime hydroxyl group of another nucleotide. However, DNA polymerases cannot synthesize DNA de novo and require a preexisting primer with a free hydroxyl group to add nucleotides and extend the chain. A specialized RNA polymerase called primase synthesizes short RNA sequences about 10 nucleotides long which serve as primers. A single primer aids DNA replication on the leading strand and multiple primers initiate okazaki fragment synthesis on the lagging strand. In Eukaryotes, the primase is part of the DNA polymerase &alpha (reviewed in [8] ). The replicative helicase and primase functionally co-operate and stimulate each other&rsquos activity [9] .

After DNA polymerase &alpha has synthesized a short, 30-40 nucleotide stretch of DNA, further DNA synthesis is handed over to polymerase &epsilon and polymerase &delta which have a higher processivity than polymerase &alpha. The higher processivity or the ability of the polymerases to stay associated with DNA for upto 10kb without falling off is due to their association with a sliding clamp called PCNA. The polymerase switching enables DNA synthesis with high fidelity as polymerase &epsilon and polymerase &delta have a 3&prime &ndash 5&prime exonuclease activity which enables proof reading and removal of any incorrect bases that is incorporated (reviewed in [8] ). At the replication fork, there is a division of labor between the polymerases where polymerase &epsilon carries out leading strand synthesis and polymerase &delta is involved in the synthesis of the lagging strand [10] , 12)


Biologists Discover a Trigger for Cell Extrusion – Process for Eliminating Unneeded Cells

For all animals, eliminating some cells is a necessary part of embryonic development. Living cells are also naturally sloughed off in mature tissues for example, the lining of the intestine turns over every few days.

One way that organisms get rid of unneeded cells is through a process called extrusion, which allows cells to be squeezed out of a layer of tissue without disrupting the layer of cells left behind. MIT biologists have now discovered that this process is triggered when cells are unable to replicate their DNA during cell division.

The researchers discovered this mechanism in the worm C. elegans, and they showed that the same process can be driven by mammalian cells they believe extrusion may serve as a way for the body to eliminate cancerous or precancerous cells.

“Cell extrusion is a mechanism of cell elimination used by organisms as diverse as sponges, insects, and humans,” says H. Robert Horvitz, the David H. Koch Professor of Biology at MIT, a member of the McGovern Institute for Brain Research and the Koch Institute for Integrative Cancer Research, a Howard Hughes Medical Institute investigator, and the senior author of the study. “The discovery that extrusion is driven by a failure in DNA replication was unexpected and offers a new way to think about and possibly intervene in certain diseases, particularly cancer.”

MIT postdoc Vivek Dwivedi is the lead author of the paper, which was published on May 5, 2021, in Nature. Other authors of the paper are King’s College London research fellow Carlos Pardo-Pastor, MIT research specialist Rita Droste, MIT postdoc Ji Na Kong, MIT graduate student Nolan Tucker, Novartis scientist and former MIT postdoc Daniel Denning, and King’s College London professor of biology Jody Rosenblatt.

Stuck in the cell cycle

In the 1980s, Horvitz was one of the first scientists to analyze a type of programmed cell suicide called apoptosis, which organisms use to eliminate cells that are no longer needed. He made his discoveries using C. elegans, a tiny nematode that contains exactly 959 cells. The developmental lineage of each cell is known, and embryonic development follows the same pattern every time. Throughout this developmental process, 1,090 cells are generated, and 131 cells undergo programmed cell suicide by apoptosis.

Horvitz’s lab later showed that if the worms were genetically mutated so that they could not eliminate cells by apoptosis, a few of those 131 cells would instead be eliminated by cell extrusion, which appears to be able to serve as a backup mechanism to apoptosis. How this extrusion process gets triggered, however, remained a mystery.

To unravel this mystery, Dwivedi performed a large-scale screen of more than 11,000 C. elegans genes. One by one, he and his colleagues knocked down the expression of each gene in worms that could not perform apoptosis. This screen allowed them to identify genes that are critical for turning on cell extrusion during development.

To the researchers’ surprise, many of the genes that turned up as necessary for extrusion were involved in the cell division cycle. These genes were primarily active during first steps of the cell cycle, which involve initiating the cell division cycle and copying the cell’s DNA.

Further experiments revealed that cells that are eventually extruded do initially enter the cell cycle and begin to replicate their DNA. However, they appear to get stuck in this phase, leading them to be extruded.

Most of the cells that end up getting extruded are unusually small, and are produced from an unequal cell division that results in one large daughter cell and one much smaller one. The researchers showed that if they interfered with the genes that control this process, so that the two daughter cells were closer to the same size, the cells that normally would have been extruded were able to successfully complete the cell cycle and were not extruded.

The researchers also showed that the failure of the very small cells to complete the cell cycle stems from a shortage of the proteins and DNA building blocks needed to copy DNA. Among other key proteins, the cells likely don’t have enough of an enzyme called LRR-1, which is critical for DNA replication. When DNA replication stalls, proteins that are responsible for detecting replication stress quickly halt cell division by inactivating a protein called CDK1. CDK1 also controls cell adhesion, so the researchers hypothesize that when CDK1 is turned off, cells lose their stickiness and detach, leading to extrusion.

Cancer protection

Horvitz’s lab then teamed up with researchers at King’s College London, led by Rosenblatt, to investigate whether the same mechanism might be used by mammalian cells. In mammals, cell extrusion plays an important role in replacing the lining of the intestines, lungs, and other organs.

The researchers used a chemical called hydroxyurea to induce DNA replication stress in canine kidney cells grown in cell culture. The treatment quadrupled the rate of extrusion, and the researchers found that the extruded cells made it into the phase of the cell cycle where DNA is replicated before being extruded. They also showed that in mammalian cells, the well-known cancer suppressor p53 is involved in initiating extrusion of cells experiencing replication stress.

That suggests that in addition to its other cancer-protective roles, p53 may help to eliminate cancerous or precancerous cells by forcing them to extrude, Dwivedi says.

“Replication stress is one of the characteristic features of cells that are precancerous or cancerous. And what this finding suggests is that the extrusion of cells that are experiencing replication stress is potentially a tumor suppressor mechanism,” he says.

The fact that cell extrusion is seen in so many animals, from sponges to mammals, led the researchers to hypothesize that it may have evolved as a very early form of cell elimination that was later supplanted by programmed cell suicide involving apoptosis.

“This cell elimination mechanism depends only on the cell cycle,” Dwivedi says. “It doesn’t require any specialized machinery like that needed for apoptosis to eliminate these cells, so what we’ve proposed is that this could be a primordial form of cell elimination. This means it may have been one of the first ways of cell elimination to come into existence, because it depends on the same process that an organism uses to generate many more cells.”

Reference: “Replication stress promotes cell elimination by extrusion” by Vivek K. Dwivedi, Carlos Pardo-Pastor, Rita Droste, Ji Na Kong, Nolan Tucker, Daniel P. Denning, Jody Rosenblatt and H. Robert Horvitz, 5 May 2021, Nature.
DOI: 10.1038/s41586-021-03526-y

Dwivedi, who earned his PhD at MIT, was a Khorana scholar before entering MIT for graduate school. This research was supported by the Howard Hughes Medical Institute and the National Institutes of Health.


How Does DNA Store Genetic Information?

DNA, also known as Deoxyribonucleic acid is the blueprint of life – meaning it stores all information that makes up any living organism. But, how does DNA store genetic information?

James Watson and Francis Crick discovered the structure of DNA – two (polynucleotide) strands intertwined in a double helix –in the year 1953. They found that DNA stores information using a simple four-letter code, which involves a cool feature known as complementary base pairing.

So how does complementary base pairing work?

We all have learned that DNA has two strands twisted around each other to form double helix, which kind of would resemble a ladder if we flatten it out.

In DNA, each step in the ladder is made of two pieces, and these two pieces are called bases. DNA has four bases named – adenine, cytosine, guanine and thymine. They are abbreviated simply by their first initials, that is – A,C, G and T – and these letters represent the code DNA uses to store genetic information.

One key part of Watson and Crick’s discovery was the way in which these bases pair up with one another. They found that A pairs only with T, and C pairs only with G. That is – A and T complement one another and C in G complement one another. This also means that C and G cannot pair with A or T.

For example, if one strand of DNA has bases CTGAC, the other will have GACTG. One way to remember which bases pair with which is to write the letters in alphabetical order, then below that – write the letters in the reverse order.

But, what does all this mean for a cell?

When a cell prepares for cell division, it makes a replica of its DNA so that the two resulting daughter cells have exactly the same genetic information, or DNA, as the parent cell. So complementary base pairing means that cells can replicate their DNA quickly and efficiently.

During the replication process, the double helix separates down the middle where the pairs of bases join. This exposes the sequences of A’s C’s G’s and T’s on each side. Then, groups of enzymes come along and add complementary bases one after another along the entire length of both DNA strands, that is – gene after gene along the entire length of the chromosome. Then in the end, there are two identical chromosomes that are encoded with the same genetic information.

Complementary base pairing also plays an important role when cells make proteins. During protein synthesis, the DNA that is the gene that codes for the needed protein – opens up, which exposes the sequences of bases in that gene. The sequence of bases is then copied in the form of RNA, but with one important difference – RNA has no T (thymine). Instead of the base thymine, it uses a base called uracil (abbreviated as U). So, when a gene is copied during protein synthesis, every A in the DNA is matched with a U in the RNA.

You can read more about how the cell converts DNA into working proteins at – Translation: DNA to mRNA to Protein (Nature Education)

Complementary base pairing has helped scientists in understanding how cancer happens. For example, if they know the sequence of bases for a piece of DNA from a cell, they can learn what the base sequence is for the opposite piece by using complementary base pairing.

Gene sequencing technology reveals the lineup of bases in DNA and a messenger RNA. Scientists can then use complementary base pairing to identify the gene that produced that messenger RNA. Once scientists know the sequence of amino acids in a protein, they can decipher the sequence of bases in the messenger RNA for the protein and then the gene that codes for that protein.

Complementary base pairing also allows scientists to compare genes from cancer cells and healthy cells from a patient. This helps them learn more about why cancer happens and how tumors grow.

Watson and Crick’s discovery has been called the most important biological work of all time. It also earned them the Nobel Prize for Physiology or Medicine in 1962.


DNA Replication

In mitosis, the cell splits apart to form two identical, same cells. That means that it has the same #"DNA"# and number of chromosomes as the previous cell. So, mitosis's main function is literally #"DNA"# replication.

Answer:

Base pair in DNA replication is a way that the chromosomes have to double check to make sure that the duplication is exact.

Explanation:

Base pair in DNA replication is a way that the chromosomes have to double check to make sure that the duplication is exact.

The replication is termed semiconservative since each new cell contains one strand of original DNA and one newly synthesized strand of DNA. The original polynucleotide strand of DNA serves as a template to guide the synthesis of the new complementary polynucleotide of DNA. A template is a guide that may be used for example, by a carpenter to cut intricate designs in wood.


Introduction

Topoisomerases unlink DNA during and after replication ( Ullsperger et al., 1995 ). Lk, the linking number of the parental DNA strands, must be reduced to zero for separation of daughter DNA molecules. ΔLk is the difference between Lk and Lk 0 , the value of Lk for the same DNA molecule in relaxed form. The unwinding of parental DNA by replicative helicases causes a compensatory (+)ΔLk that must be removed by topoisomerases. Champoux and Been (1980) suggested that the (+)ΔLk could take the form of (+) supercoils in the unreplicated region as well as windings of the two newly replicated regions around each other. Thus, topoisomerases could unlink replication intermediates (RIs) by acting either in front of or behind the fork. The form of (+)ΔLk in the replicated DNA was subsequently named precatenane, due to structural similarity to catenanes and to distinguish it from the (+)ΔLk in the unreplicated DNA ( Ullsperger et al., 1995 ). Any (+)ΔLk not removed before termination would form catenanes, and these would be unlinked after replication.

This model did not achieve immediate acceptance because early electron microscopy (EM) observations of circular RIs showed supercoils but no precatenanes, suggesting that topoisomerases only act ahead of the fork. However, studies of plasmid replication with purified Escherichia coli enzymes suggested that precatenanes are important in unlinking during replication ( Peng and Marians, 1993 Hiasa and Marians, 1994 , 1996 ). Moreover, both (−) precatenanes and (−) supercoils were observed on purified RIs accumulated by replication of plasmids containing two termination sites in E.coli ( Peter et al., 1998 ). An EM artefact apparently caused earlier studies to miss precatenanes. Finally, a topological analysis of knots trapped within arrested RIs suggested that (−) precatenanes exist in E.coli cells ( Sogo et al., 1999 ).

However, removing (−) precatenanes would just increase Lk. Arrested RIs from E.coli cells have a (−)ΔLk, presumably due to gyrase activity after replication arrest. Direct evidence for precatenanes on (+)ΔLk RIs, as predicted by Champoux and Been, has been lacking. In fact, (+)ΔLk RIs prepared by adding intercalating agents to purified (−)ΔLk RIs contain neither supercoils nor precatenanes. Instead, the (+) topological stress is relieved by re-annealing of the parental strands and formation of a Holliday junction, a process called fork reversal ( Postow et al., 2001 J.B.Schvartzman, personal communication). It remains unclear, at least in bacteria, whether transient (not arrested) RIs carry a (+) or a (−)ΔLk and whether protein binding inside the cell prevents fork reversal and/or spinning and (+) precatenane formation.

In bacteria, two type 2 topoisomerases can unlink replicating DNA. Gyrase introduces (−) supercoils in front of the forks and may suffice to overcome the (+)ΔLk generated by replication until late RI stages, while topoisomerase IV (topo IV) is responsible for decatenating complete replication products (reviewed in Levine et al., 1998 ). Studies with purified enzymes support a role for precatenane unlinking by topo IV ( Peng and Marians, 1993 Hiasa and Marians, 1996 ). However, in topo IV mutants, newly synthesized plasmid DNA accumulates as catenanes with the same node number distribution as transient catenanes in wild-type cells ( Zechiedrich and Cozzarelli, 1995 ). Thus, in vivo evidence for precatenane removal by topo IV is lacking. In fact, the recent discovery that topo IV relaxes (+) supercoils 20-fold faster than (−) supercoils suggests that it may unlink DNA in front of the fork as efficiently as gyrase ( Crisona et al., 2000 ).

Eukaryotes lack unconstrained (−) supercoils and the (−) supercoiling activity of gyrase. Thus, free (+) supercoils and possibly (+) precatenanes are expected to form during elongation. Both topo I and topo II can remove (+) supercoils in vitro. Studies with yeast mutants showed that replication can initiate in the absence of both enzymes, but elongation stops after a couple of thousand base pairs ( Kim and Wang, 1989 ). Either topo I or topo II (but not topo III) can support further elongation and completion of S phase ( Uemura and Yanagida, 1986 Brill et al., 1987 ). Topo II is strictly required at mitosis for separation of sister chromatids ( Holm et al., 1985 Uemura and Yanagida, 1986 ). Similar observations were made for the replication of naked SV40 DNA in cell-free extracts ( Yang et al., 1987 ) or with purified proteins ( Ishimi et al., 1992b ). The usual interpretation is that either topo I or topo II can remove (+) supercoils to drive elongation, while topo II is required to remove catenane crossings persisting after replication. However, it is unclear whether topo II relaxes (+) supercoils in front of the forks, like topo I, or removes (+) precatenanes behind the forks. Studies of SV40 minichromosome replication in cellular extracts or in infected cells suggest that topo II inhibition in some cases blocks elongation at the late RI stage ( Richter et al., 1987 Ishimi et al., 1992a , b ), but in other cases allows synthesis of complete catenated dimers, even though late RIs accumulate ( Sundin and Varshavsky, 1981 Ishimi et al., 1995 and references therein reviewed in Snapka, 1996 ). Thus, the implication of topo II in the late stages of DNA synthesis in higher eukaryotes is unclear.

Another unresolved issue is what drives decatenation of daughter DNA molecules. In vitro, topo II catalyses both catenation and decatenation of DNA rings, and favours catenation at high DNA concentration ( Krasnow and Cozzarelli, 1982 ). Given the high concentration of DNA in vivo, one expects the equilibrium to be toward catenation. Mechanical separation of sister chromatids during mitosis has been proposed to drive decatenation ( Holm, 1994 Duplantier et al., 1995 ). Although experiments in yeast and Xenopus show that topo II is required for mitotic chromosome condensation and segregation (reviewed in Holm, 1994 ), it is not known whether decatenation is postponed entirely until mitosis or already starts in S or G2 phases.

To investigate these questions, we have studied the effect of topo II inhibition on DNA replication in Xenopus egg extracts. Because studying replication and topology of a long linear chromosome would be difficult, we focused on circular plasmid DNA. Any plasmid DNA incubated in Xenopus egg extracts is replicated under cell cycle control, but only after it has been assembled by the egg extract into chromatin and then into synthetic nuclei, in which replication occurs at discrete foci as in normal nuclei ( Blow and Laskey, 1986 Blow and Sleeman, 1990 Cox and Laskey, 1991 ). Small plasmids (<15 kb) support a single, randomly located initiation event that closely mimics replication of chromosomal domains in early embryonic nuclei ( Hyrien and Méchali, 1992 , 1993 Mahbubani et al., 1992 Lucas et al., 2000 ). Although caution is required because plasmids may be free of some of the topological restraints of long linear chromosomes, extrapolation from this system to what happens inside cells seems reasonable.

We have analysed the effect of various topo II inhibitors on plasmid DNA replication using high-resolution two-dimensional gel electrophoresis of replication products. ICRF-193 traps the enzyme in the form of a closed protein clamp without introducing DNA breaks ( Tanabe et al., 1991 ). Etoposide (VP-16) and teniposide (VM-26) poison topo II by stabilizing a covalent reaction intermediate, the cleavable complex. Subsequent treatment with protein denaturants reveals the DNA strand breaks and the covalent linking of a topoisomerase subunit to the 5′ end of the broken DNA ( Chen et al., 1984 ). The covalent intermediates can be extracted selectively with phenol prior to deproteinization. These properties were exploited to map the sites of topo II action during DNA replication. Our results provide direct evidence for the Champoux and Been model in this eukaryotic system, and reveal a division of labour between topo I and topo II. We suggest a role for chromatin assembly in driving DNA unlinking behind the replication fork.


Biology 171

By the end of this section, you will be able to do the following:

  • Explain how the structure of DNA reveals the replication process
  • Describe the Meselson and Stahl experiments

The elucidation of the structure of the double helix provided a hint as to how DNA divides and makes copies of itself. In their 1953 paper, Watson and Crick penned an incredible understatement: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” With specific base pairs, the sequence of one DNA strand can be predicted from its complement. The double-helix model suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. What was not clear was how the replication took place. There were three models suggested ((Figure)): conservative, semi-conservative, and dispersive.


In conservative replication, the parental DNA remains together, and the newly formed daughter strands are together. The semi-conservative method suggests that each of the two parental DNA strands acts as a template for new DNA to be synthesized after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. In the dispersive model, both copies of DNA have double-stranded segments of parental DNA and newly synthesized DNA interspersed.

Meselson and Stahl were interested in understanding how DNA replicates. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen ( 15 N), which gets incorporated into nitrogenous bases, and eventually into the DNA ((Figure)).


The E. coli culture was then placed into medium containing 14 N and allowed to grow for several generations. After each of the first few generations, the cells were harvested and the DNA was isolated, then centrifuged at high speeds in an ultracentrifuge. During the centrifugation, the DNA was loaded into a gradient (typically a solution of salt such as cesium chloride or sucrose) and spun at high speeds of 50,000 to 60,000 rpm. Under these circumstances, the DNA will form a band according to its buoyant density: the density within the gradient at which it floats. DNA grown in 15 N will form a band at a higher density position (i.e., farther down the centrifuge tube) than that grown in 14 N. Meselson and Stahl noted that after one generation of growth in 14 N after they had been shifted from 15 N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15 N and 14 N. This suggested either a semi-conservative or dispersive mode of replication. The DNA harvested from cells grown for two generations in 14 N formed two bands: one DNA band was at the intermediate position between 15 N and 14 N, and the other corresponded to the band of 14 N DNA. These results could only be explained if DNA replicates in a semi-conservative manner. And for this reason, therefore, the other two models were ruled out.

During DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strands will be complementary to the parental or “old” strands. When two daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter cells.

Click through DNA Replication (Flash animation).

Section Summary

During cell division, each daughter cell receives a copy of each molecule of DNA by a process known as DNA replication. The single chromosome of a prokaryote or each chromosome of a eukaryote consists of a single continuous double helix. The model for DNA replication suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. In the conservative model of replication, the parental DNA is conserved, and the daughter DNA is newly synthesized. The semi-conservative model suggests that each of the two parental DNA strands acts as template for new DNA to be synthesized after replication, each double-stranded DNA retains the parental or “old” strand and one “new” strand. The dispersive model suggested that the two copies of the DNA would have segments of parental DNA and newly synthesized DNA. The Meselson and Stahl experiment supported the semi-conservative model of replication, in which an entire replicated chromosome consists of one parental strand and one newly synthesized strand of DNA.

Free Response

How did the scientific community learn that DNA replication takes place in a semi-conservative fashion?

Meselson’s experiments with E. coli grown in 15 N deduced this finding.

Imagine the Meselson and Stahl experiments had supported conservative replication instead of semi-conservative replication. What results would you predict to observe after two rounds of replication? Be specific regarding percent distributions of DNA incorporating 15 N and 14 N in the gradient.

Following two rounds of conservative replication, two bands would be detected after ultracentrifugation. A lower (heavier) band would be at the 15 N density, and would comprise 25% of the total DNA. A second, higher (lighter) band would be at the 14 N density, and would contain 75% of the total DNA.


DNA Replication

Since DNA forms the genetic code and that it is known that genes may be inherited, it follows that DNA must be copied exactly before being incorporated into gametes at meiosis.

It also follows that all new cells in an organism must gain a copy of the genes at mitosis, because they are able to continue the characteristic biochemical behaviour of that organism.

What is the mechanism for this exact copying or replication of the DNA?

Three theories existed:

  1. The parent DNA molecule breaks into segments and new nucleotides fill in the gaps precisely (fragmentation theory).
  2. The complete parent DNA molecule acts as a template for the new daughter molecule, which is assembled from new nucleotides. The parent molecule is unchanged (conservative hypothesis).
  3. The parent DNA molecule separates into its two component strands, each of which acts as a template for the formation of a new complementary strand. The two daughter molecules therefore contain half the parent DNA and half new DNA (semi-conservative hypothesis).

The semi conservative hypothesis

The semi conservative hypothesis was shown to be the true mechanism by the work of Meselsohn and Stahl (1958).

In their experiment they grew the bacterium E.coli in the presence of radioactive 15 N until a culture was obtained in which all the DNA was labelled with 15 N.

A subculture of this labelled bacterium was than transferred for growth in the presence of normal 14 N. The generation time of E.coli is known, so it was possible to take samples of this growing subculture after exactly one, two, three generations and so on.

Each sample had its DNA extracted and the isolated DNA was then centrifuged in a caesium chloride solution (high viscosity) at 40,000G for 20 hours, causing the DNA to sediment out.

The heavier the DNA, the further it moved down the centrifuge tubes. 15 N DNA is heavier than 14 N DNA. Mixed 14 N and 15 N DNA is intermediate in mass between the two.

The original 15 N DNA moved to the lowest position in the tube.

After one generation all the DNA moved to an intermediate position, indicating the presence of only mixed 14 N and 15 N DNA. This was because the DNA in this generation contained one strand of the parent molecule and one new 14 N strand.

Had the conservative hypothesis been true, two DNA masses would have been visible, one heavy and the other light.

In the second generation half the DNA was intermediate and half was light, for the same reason.

Process of DNA replication

The actual process is simple. To begin with one strand in the DNA duplex is nicked by the enzyme DNA topoisomerase, allowing part of the molecule to unravel to form a replication fork (the DNA is replicated a bit at a time and the whole molecule is never completely uncoiled).

Next, the enzyme DNA helicase splits the two strands by breaking the hydrogen bonds. This exposes the bases.

DNA polymerase enzyme then moves along the exposed bases sequences, creating a new complementary strand as it goes. DNA polymerase reads the exposed code from the 3' to the 5' end and therefore assembles the new strand from the 5' to the 3'.

Several molecules of DNA polymerase act simultaneously, each assembling a separate section of the new strand of DNA. Each DNA polymerase is preceded by an RNA polymerase enzyme, which constructs an RNA primer to guide the action of the DNA polymerase.

These new DNA segments are then joined together by the enzyme DNA ligase. The two new daughter molecules then coil up again to reform the double helix structure. This process occurs during the S phase of the cell cycle.