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When hydrogen bonds happen between the RNA nucleotide bases and the DNA bases , do phosphodiester bonds form between the RNA nucleotides in the primer ? No source I read is clear about this, are their bonds between the RNA nucleotides ?
A primer is by definition a short single strand of nucleic acid (i.e. a series of nucleotides linked together by phosphodiester bonds). See also this wikipedia article.
DNA replication is a biological process that occurs in all living organisms acting as the most essential part of biological inheritance. It is the process by which DNA makes a copy of itself during cell division.
Rules of DNA replication in eukaryotes
- DNA replication is semiconservative: Each DNA strand serves as a template for synthesis of a new strand producing two DNA molecules, each with one new strand and one old strand.
- Replication begins at multiple origins and usually proceeds bidirectionally. Having multiple origins of replication provides a mechanism for rapidly replicating the great length of eukaryotic DNA molecules.
- Replication exhibits polarity: DNA synthesis proceeds in a 5´ → 3´ direction and is semi discontinuous.
- Replication is very accurate: replication proceeds with an extraordinary degree of fidelity.
- DNA replication occurs in the nucleus during the synthetic (S) phase of the eukaryotic cell cycle. This phase is preceded and followed by two periods during which DNA is not synthesized (gap periods G1, and G2). During cell division (mitotic M phase), each daughter cell receives one of the two identical DNA molecules.
During polymerization, discrimination between correct and incorrect nucleotides depends on base-pairing interactions (A=T and G≡C). Proofreading 3´→ 5´ exonuclease activity double- checks each nucleotide after it is added and removes the mispaired one. The additional accuracy is accounted for by a separate enzyme system that repairs the mismatched base pairs remaining after replication.
Requirements of DNA replication in eukaryotes
DNA is synthesized by DNA polymerases
DNA polymerases require the presence of a primer (i.e. oligonucleotide of RNA with free 3´ hydroxyl group), a template (i.e single-stranded DNA), and deoxyribonucleotides (d ATP, d CTP, d GTP, and d TTP) in order to function. The primer provides a site for the polymerization to begin. The free 3´ hydroxyl group of the primer acts as an accepter for the first deoxyribonucleotide in the newly formed DNA strand.
DNA polymerases utilize one deoxyribonucleoside triphosphate as a source of the deoxyribonucleoside monophosphate for the growing DNA strand by the removal of pyrophosphate. The selection of the incoming deoxyribonucleotide is dependent upon proper base pairing with the template. The RNA-primed synthesis of DNA demonstrating the template function of the complementary strand parental DNA.
In eukaryotes, there are at least 5 DNA polymerase enzymes (α, β, γ, δ and ε )
- DNA polymerase α has a Primase activity (for the synthesis of RNA primer)Polymerization activity (formation of phosphodiester bond), andNo proofreading 3´ → 5´ exonuclease activity.
- DNA polymerase β functions in DNA repair (it has 5´→ 3´ exonuclease activity).
- DNA polymerase γ synthesizes mitochondrial DNA.
- DNA polymerase δ has Polymerization activity and Proofreading 3´→5´ exonuclease activity.
- DNA polymerase ε removes the primers of Okazaki fragments on the lagging strand. It has DNA repair and proofreading activities.
DNA is degraded by nucleases or DNases
They may be either exonucleases or endonucleases. Exonucleases degrade nucleic acids from one end of the molecule operating either from 5´→ 3´ or from 3´ →5´ direction of one strand of the double-stranded nucleic acids.
Endonucleases can begin to degrade at any internal site in a nucleic acid strand, reducing it to smaller and smaller fragments. Other enzymes (e.g. helicase, topoisomerase, and DNA ligase) and protein factors (e.g. origin binding proteins and single-stranded binding proteins) are required for the replication process.
Steps of DNA replication in eukaryotes
The synthesis of a DNA molecule can be divided into three stages: initiation, elongation, and termination.
Identification of the origins of replication: Origins of replication in eukaryotes (e.g. yeast) are called replicators. These origins are located adjacent to A-T- rich sequence that is easy to unwind. Origin recognition complex (ORC) is a set of proteins that can bind to these replicators.
The unwinding of double-stranded DNA: The interaction of these proteins with the replicators leads to the unwinding of DNA forming “replication bubbles”.
Formation of replication forks:
- When the two DNA strands unwind and separate, they form two replication forks at each origin. Replication of double-stranded DNA is bidirectional i.e. replication forks move in both directions away from the origin.
- DNA helicase binds to the unwound region near the replication fork and then move into the neighboring double-stranded region, forcing the strands apart. Helicase uses energy from ATP to break the hydrogen bonds holding the base pairs together.
- As helicase unwinds the DNA at the replication forks, the DNA ahead of it becomes overwound and positive supercoils forms. Topoisomerase II removes these supercoils (by nicking both strands of DNA, passing the DNA strands through the nick, and then resealing both strands again).
- Eukaryotic single-stranded DNA binding protein binds to the single-stranded portion of each DNA strand, preventing the strands from reassociation and protecting them from degradation by nucleases.
RNA primer synthesis: DNA polymerase α with primase activity is responsible for the synthesis of a short RNA primer in 5´→ 3´ direction, beginning at the origin of each parental strand. The parental strand is used as a template for this process.
The elongation phase of replication includes 2 distinct but related operations: leading and lagging strand synthesis.
Leading strand synthesis begins with the synthesis of RNA primer by DNA polymerase α at the replication origin. Deoxyribonucleotides are added to this primer by DNA polymerase δ. Leading strand synthesis then proceeds continuously, in the direction of replication fork movement.
Lagging strand synthesis is synthesized discontinuously in the opposite direction from the replication fork movement as a series of small fragments known as Okazaki fragments. Each Okazaki fragment is initiated by the synthesis of an RNA primer by polymerase α and then completed by the synthesis of DNA by DNA polymerase δ until reaching the next RNA primer.
Both leading and Okazaki fragments of lagging strands are synthesized from 5´ → 3´ direction. There is a leading and a lagging strand for each of the two replication forks. Then, DNA polymerase ε begins to remove the RNA primers. The gap is then filled by a polymerase (δ/ε).
Both DNA polymerase δ and ε have the ability to proofread their work by means of a 3´→5´ exonuclease activity. If DNA polymerase makes a mistake during DNA synthesis, the resulting unpaired base at the 3´ end of the growing strand is removed before synthesis continues.
Ligation of the newly synthesized DNA segments: DNA ligase seals the nicks between Okazaki fragments, converting them to a continuous strand of DNA. DNA ligase requires ATP to form a phosphodiester bond between the two nucleotides.
The termination of replication on linear eukaryotic chromosomes involves the synthesis of telomeres at the ends of the chromosome.
They are repetitive sequences TTAGGGs, these telomeres (Hexamers) cap the end of human chromosomes and are believed to prevent chromosomes from undergoing degradation. Telomeres are believed to contribute to the preservation of the normal aging process. With each round of replication in most normal cells, the telomeres are shortened.
During normal cell division, the length of telomeres is shortened, thus limiting the life span of the cell, Therefore, normal somatic cells after a certain number of cell division die due to the shortening of telomeres. So telomeres determine the proliferation capacity of human somatic cells.
It is an enzyme in eukaryotes used to maintain the telomeres. It contains a short RNA template complementary to the DNA telomere sequence, as well as telomerase reverse transcriptase activity. Telomerase is thus able to replace telomere sequences that would otherwise be lost during replication. Normally, telomerase activity is present only in embryonic cells, germ (reproductive) cells, and stem cells but not in somatic cells.
Cancer cells often have relatively high levels of telomerase, preventing the telomeres from becoming shortened and contributing to the immortality of malignant cells. After replication is complete, the parent and daughter strands reform double-stranded DNA.
In eukaryotic cells, the double-stranded DNA must precisely reform the chromatin structure including nucleosomes that existed prior to the onset of replication. The 2 identical sister chromatids are separated from each other when the cell divides during mitosis.
Quinolones are a family of drugs that block the action of prokaryotic topoisomerase thus preventing DNA replication and transcription. These drugs such as Nalidixic acid and ciprofloxacin act as antibiotics. While inhibitors of eukaryotic topoisomerases as Etoposide are becoming useful as anticancer agents.
A. Visualizing Replication and Replication Forks
Recall the phenomenon of bacterial conjugation allowed a demonstration bacterial chromosomes were circular. In 1963, John Cairns confirmed this fact by direct visualization of bacterial DNA. He cultured E. coli cells for long periods on 3Hthymidine (3H-T) to make all of their cellular DNA radioactive. He then disrupted the cells gently to minimize damage to the DNA. The DNA released was allowed to settle and adhere to membranes. A sensitive film was placed over the membrane and time was allowed for the radiation to expose the film. After Cairns developed the autoradiographs, he examined the results in the electron microscope. He saw tracks of silver grains in the autoradiographs (the same kind of silver grains that create an image on film in old-fashioned photography). Look at the two drawings of his autoradiographs on the next page.
Cairns measured the length of the &ldquosilver&rdquo tracks, which usually consisted of three possible closed loops, or circles. The circumferences of two of these circles were always equal, their length closely predicted by the DNA content of a single, nondividing cell. Cairns therefore interpreted these images to be bacterial DNA in the process of replication. Cairns&rsquo autoradiographs and the measurements that led him to conclude that he was looking at images of bacterial circular chromosomes are illustrated below.
He arranged his autoradiograph images in a sequence (below) to make his point.
Because the replicating chromosomes looked (vaguely!) like the Greek letter ( heta ), Cairns called them theta images. He inferred that replication starts at a single origin of replication on the bacterial chromosome, proceeding around the circle to completion.
Subsequent experiments by David Prescott demonstrated bidirectional replication&hellip, that replication did indeed begin at an origin of replication, after which the double helix was unwound and replicated in both directions, away from the origins, forming two replication forks (illustrated below).
Bacterial cells can divide every hour (or even less) the rate of bacterial DNA synthesis is about 2 X 106 base pairs per hour. A typical eukaryotic cell nucleus contains thousands of times as much DNA as a bacterium, and typical eukaryotic cells double every 15-20 hours. Even a small chromosome can contain hundreds or thousands of times as much DNA as a bacterium. It appeared that eukaryotic cells could not afford to double their DNA at a bacterial rate of replication! Eukaryotes solved this problem not by evolving a faster biochemistry of replication, but by using multiple origins of replication from which DNA synthesis proceeds in both directions. This results in the creation of multiple replicons.
Each replicon enlarges, eventually meeting other growing replicons on either side to replicate most of each linear chromosome, suggested in the illustration below.
Before we consider the biochemical events at replication forks in detail, let's look at the role of DNA polymerase enzymes in the process.
In this chapter (Enzymes Involved in DNA Replication) and the next, we will examine the process of replication.
After describing the basic mechanism of DNA replication, we discuss the various techniques researchers have used to achieve a complete understanding of replication.
Indeed, a theme of this chapter is the combination of genetic and biochemical approaches that have allowed us to uncover the mechanism and physiology of DNA replication.
Enzymes known as DNA polymerases are responsible creating the new strand by a process called elongation. There are five different known types of DNA polymerases in bacteria and human cells. In bacteria such as E. coli, polymerase III is the main replication enzyme, while polymerase I, II, IV and V are responsible for error checking and repair. DNA polymerase III binds to the strand at the site of the primer and begins adding new base pairs complementary to the strand during replication. In eukaryotic cells, polymerases alpha, delta, and epsilon are the primary polymerases involved in DNA replication. Because replication proceeds in the 5' to 3' direction on the leading strand, the newly formed strand is continuous.
The lagging strand begins replication by binding with multiple primers. Each primer is only several bases apart. DNA polymerase then adds pieces of DNA, called Okazaki fragments, to the strand between primers. This process of replication is discontinuous as the newly created fragments are disjointed.
14.1 | Historical Basis of Modern Understanding
By the end of this section, you will be able to:
- Explain transformation of DNA.
- Describe the key experiments that helped identify that DNA is the genetic material.
- State and explain Chargaff’s rules
Modern understandings of DNA have evolved from the discovery of nucleic acids to the development of the double-helix model. In the 1860s, Friedrich Miescher (Figure 14.2), a physician by profession, was the first person to isolate phosphate- rich chemicals from white blood cells or leukocytes. He named these chemicals (which would eventually be known as RNA and DNA) nuclein because they were isolated from the nuclei of the cells.
Figure 14.2 Friedrich Miescher (1844–1895) discovered nucleic acids.
A half century later, British bacteriologist Frederick Griffith was perhaps the first person to show that hereditary information could be transferred from one cell to another “horizontally,” rather than by descent. In 1928, he reported the first demonstration of bacterial transformation, a process in which external DNA is taken up by a cell, thereby changing morphology and physiology. He was working with Streptococcus pneumoniae, the bacterium that causes pneumonia. Griffith worked with two strains, rough (R) and smooth (S). The R strain is non-pathogenic (does not cause disease) and is called rough because its outer surface is a cell wall and lacks a capsule as a result, the cell surface appears uneven under the microscope. The S strain is pathogenic (disease-causing) and has a capsule outside its cell wall. As a result, it has a smooth appearance under the microscope. Griffith injected the live R strain into mice and they survived. In another experiment, when he injected mice with the heat-killed S strain, they also survived. In a third set of experiments, a mixture of live R strain and heat-killed S strain were injected into mice, and—to his surprise—the mice died. Upon isolating the live bacteria from the dead mouse, only the S strain of bacteria was recovered. When this isolated S strain was injected into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S strain into the live R strain and transformed it into the pathogenic S strain, and he called this the transforming principle (Figure 11.3). These experiments are now famously known as Griffith’s transformation experiments.
Figure 14.3 Two strains of S.pneumoniae were used in Griffith’s transformation experiments. The R strain is non- pathogenic. The S strain is pathogenic and causes death. When Griffith injected a mouse with the heat-killed S strain and a live R strain, the mouse died. The S strain was recovered from the dead mouse. Thus, Griffith concluded that something had passed from the heat-killed S strain to the R strain, transforming the R strain into S strain in the process. (credit “living mouse”: modification of work by NIH credit “dead mouse”: modification of work by Sarah Marriage)
Scientists Oswald Avery, Colin MacLeod, and Maclyn McCarty (1944) were interested in exploring this transforming principle further. They isolated the S strain from the dead mice and isolated the proteins and nucleic acids, namely RNA and DNA, as these were possible candidates for the molecule of heredity. They conducted a systematic elimination study. They used enzymes that specifically degraded each component and then used each mixture separately to transform the R strain. They found that when DNA was degraded, the resulting mixture was no longer able to transform the bacteria, whereas all of the other combinations were able to transform the bacteria. This led them to conclude that DNA was the transforming principle.
Experiments conducted by Martha Chase and Alfred Hershey in 1952 provided confirmatory evidence that DNA was the genetic material and not proteins. Chase and Hershey were studying a bacteriophage, which is a virus that infects bacteria. Viruses typically have a simple structure: a protein coat, called the capsid, and a nucleic acid core that contains the genetic material, either DNA or RNA. The bacteriophage infects the host bacterial cell by attaching to its surface, and then it injects its nucleic acids inside the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a large number of bacteriophages. Hershey and Chase labeled one batch of phage with radioactive sulfur, 35 S, to label the protein coat. Another batch of phage were labeled with radioactive phosphorus, 32 P. Because phosphorous is found in DNA, but not protein, the DNA and not the protein would be tagged with radioactive phosphorus.
Each batch of phage was allowed to infect the cells separately. After infection, the phage bacterial suspension was put in a blender, which caused the phage coat to be detached from the host cell. The phage and bacterial suspension was spun down in a centrifuge. The heavier bacterial cells settled down and formed a pellet, whereas the lighter phage particles stayed in the supernatant (the liquid above the pellet). In the tube that contained phage labeled with 35 S, the supernatant contained the radioactively labeled phage, whereas no radioactivity was detected in the pellet. In the tube that contained the phage labeled with 32 P, the radioactivity was detected in the pellet that contained the heavier bacterial cells, and no radioactivity was detected in the supernatant. Hershey and Chase concluded that it was the phage DNA that was injected into the cell and carried information to produce more phage particles, thus providing evidence that DNA was the genetic material and not proteins (Figure 14.4).
Figure 14.4 In Hershey and Chase’s experiments, bacteria were infected with phage radiolabeled with either 35S, which labels protein, or 32P, which labels DNA. Only 32P entered the bacterial cells, indicating that DNA is the genetic material.
Around this same time, Austrian biochemist Erwin Chargaff examined the content of DNA in different species and found that the amounts of adenine, thymine, guanine, and cytosine were not found in equal quantities, and that it varied from species to species, but not between individuals of the same species. He found that the amount of adenine equals the amount of thymine, and the amount of cytosine equals the amount of guanine, or A = T and G = C. These are also known as Chargaff’s rules. This finding proved immensely useful when Watson and Crick were getting ready to propose their DNA double helix model, discussed in Chapter 5.
16.5. DNA Replication in Eukaryotes
Eukaryotic genomes are much more complex and larger in size than prokaryotic genomes. The human genome has three billion base pairs per haploid set of chromosomes, and 6 billion base pairs are replicated during the S phase of the cell cycle. There are multiple origins of replication on the eukaryotic chromosome humans can have up to 100,000 origins of replication. The rate of replication is approximately 100 nucleotides per second, much slower than prokaryotic replication. In yeast, which is a eukaryote, special sequences known as Autonomously Replicating Sequences (ARS) are found on the chromosomes. These are equivalent to the origin of replication in E. coli .
The number of DNA polymerases in eukaryotes is much more than prokaryotes: 14 are known, of which five are known to have major roles during replication and have been well studied. They are known as pol α , pol β , pol γ , pol δ , and pol ε .
The essential steps of replication are the same as in prokaryotes. Before replication can start, the DNA has to be made available as template. Eukaryotic DNA is bound to basic proteins known as histones to form structures called nucleosomes. The chromatin (the complex between DNA and proteins) may undergo some chemical modifications, so that the DNA may be able to slide off the proteins or be accessible to the enzymes of the DNA replication machinery. At the origin of replication, a pre-replication complex is made with other initiator proteins. Other proteins are then recruited start the replication process.
A helicase using the energy from ATP hydrolysis opens up the DNA helix. Replication forks are formed at each replication origin as the DNA unwinds. The opening of the double helix causes over-winding, or supercoiling, in the DNA ahead of the replication fork. These are resolved with the action of topoisomerases. Primers are formed by the enzyme primase, and using the primer, DNA pol can start synthesis. While the leading strand is continuously synthesized by the enzyme pol δ , the lagging strand is synthesized by pol ε . A sliding clamp protein known as PCNA (Proliferating Cell Nuclear Antigen) holds the DNA pol in place so that it does not slide off the DNA. RNase H removes the RNA primer, which is then replaced with DNA nucleotides. The Okazaki fragments in the lagging strand are joined together after the replacement of the RNA primers with DNA. The gaps that remain are sealed by DNA ligase, which forms the phosphodiester bond (Figure 16.15).
Figure 16.15. Eukaryotic DNA replication is similar to that of baterial prokaryotes.
Unlike prokaryotic chromosomes, eukaryotic chromosomes are linear. As you’ve learned, the enzyme DNA pol can add nucleotides only in the 5′ to 3′ direction. In the leading strand, synthesis continues until the end of the chromosome is reached. On the lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer. When the replication fork reaches the end of the linear chromosome, there is no place for a primer to be made for the DNA fragment to be copied at the end of the chromosome. These ends thus remain unpaired, and over time these ends may get progressively shorter as cells continue to divide.
Figure 16.16. The telomeres, colorized pink in this electronmicrograph, are non-gene repetative sequences at the ends of chromosomes. Telomeres are sometimes called the DNA’s aglets after the plastic ends of shoe laces since they both have a protective function.
The ends of the linear chromosomes are known as telomeres (Figure 16.16), which have repetitive sequences that code for no particular gene. In a way, these telomeres protect the genes from getting deleted as cells continue to divide. In humans, a six base pair sequence, TTAGGG, is repeated 100 to 1000 times. The discovery of the enzyme telomerase (Figure 16.17) helped in the understanding of how chromosome ends are maintained. The telomerase enzyme contains a catalytic part and a built-in RNA template. It attaches to the end of the chromosome, and complementary bases to the RNA template are added on the 3′ end of the DNA strand. Once the 3′ end of the lagging strand template is sufficiently elongated, DNA polymerase can add the nucleotides complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.
Telomerase is typically active in germ cells and adult stem cells. It is not active in adult somatic cells. For her discovery of telomerase and its action, Elizabeth Blackburn (Figure 16.18) received the Nobel Prize for Medicine and Physiology in 2009.
F) L8 DNA Replication
➢ -ve supercoiling is achieved in eukaryotes from assembling of nucleosomes due to histones present - no free rotation
➢ +ve supercoiling elsewhere on DNA compensates for the -ve supercoiling to produce = 0 net change in supercoil
1. DNA replication requires exposure of template strands, DNA is unwound and create positive supercoils ahead of replication fork - must be relaxed for DNA replication to continue
2. Type 1 & 2 Topoisomerase relax negatively or positively supercoiled region and insert more negative supercoils
1) Gyrase cuts both strands of prokaryotic DNA to form a gap
2) It transfers other strand to the front then reseals the cut to form a negative supercoil - now ready for DNA replication
DNA in eukaryotes is bound by histones so also has no free rotation
When DNA is separated for DNA replication, it must be rotated to unwind it - form single strands to act as templates for DNA replication to occur
This causes over winding = generates positive supercoils ahead of replication fork - tightening helix and preventing further separation due to the tension
Prokaryotes (closed circular DNA)
Eukaryotes (DNA associated with histones)
DNA separation occurs before DNA replication - unwinding of DNA to expose template strand causes it to positively supercoil ahead of the replication fork
Type II Topoisomerases = breaks TWO strands of DNA - cuts both strands and then reseals to CREATE A NEGATIVE SUPERCOIL
1. Initiator proteins recognise origin of replication region
2. DNA HELICASE enzymes breaks H-bonds and unwinds double stranded DNA of that region
2. HELICASE enzyme (DnaB) attaches to each replication fork
= unwinds both strands by breaking H- bonds
ATP hydrolysis from Helicase releases energy to break H-bonds to unwind
3. Single stranded binding protein SSBs are added
= prevent single strands from reannealing
2 forks move in opposite directions
6. DNA synthesis : Simultaneous replication of both parent strands
LEADING STRAND (towards replication fork)
→ DNA replication occurs continuously
→ Free 3' -OH end initially on the RNA primer
= Only ONE primer is required
LAGGING STRAND (away from replication fork)
→ DNA replication occurs discontinuously
→ New RNA primers are constantly being synthesized at moving replication fork to ensure a free 3'OH end for elongation
= MORE THAN ONE primer is required
5'→3' DNA Polymerase III activity catalyzes formation of phosphodiester bonds
1. PRIMASE adds RNA Primers in several places along new growing DNA strand in the 5' → 3'
Each DNA segment requires its own RNA primer
2. DNA polymerase III adds DNA nucleotides between them discontinuously forming DNA portions until it reaches another RNA primer where it stops
= Okazaki fragments are also formed in 5' → 3' direction
3. DNA polymerase I replaces RNA primer bases with DNA nucleotides
[ 5' → 3' Polymerase activity ]
= Filling gaps between Okazaki fragments
DNA polymerases of both lagging and leading strand work in SAME DIRECTION, 5' → 3' due to looped nature of lagging strand
RNA primers are short sequence of 10 RNA nucleotides complementary to template strand that provides 3' end for Polymerase III to start on growing new DNA
(Primase is basically a RNA polymerase!)
Once the RNA primer is added, DNA polymerase "extends" 3' end of new DNA by adding nucleotides complementary to template strand
➢ Adds nucleotide bases to their complementary bases on template strand after RNA primer sequence
= Catalyses formation of phosphodiester bonds
Lagging strand : Elongates until it reaches another RNA primer of a Okazaki fragment on lagging strand
Leading stand : Elongates entire new DNA stand with only ONE INITIAL PRIMER
➢ DNA POLYMERASE CANNOT INTIATE A POLYNUCLEOTIDE CHAIN - cannot join together 2 free nucleotides
= NEEDS A PRIMER
➢ Polymerase III requires a sliding clamp
➢ DNA polymerase can only add nucleotides to 3' end of a nucleotide on pre-existing chain so extends DNA in 5' → 3' direction
[ 5' → 3' Polymerase activity ]
= DNA polymerase moves along template strand in a 3' → 5' direction, and the daughter strand is formed in a 5' → 3' direction
Back of polymerase I adds DNA nucleotides to fill gaps between Okazaki fragments towards 3' end of DNA
= 5' → 3' POLYMERASE activity
DNA polymerase I replaces RNA primers with Deoxynucleotides between the Okazaki fragments gaps
To complete the strand, DNA Ligase links the nicks together - forms phosphodiester bond
DNA polymerase binds the 5' end of new nucleotide on to the 3' end of RNA primer of new growing DNA strand
[ A DNA double helix is anti-parallel ]
5' → 3' continuous ✔✔
2 sets of DNA polymerases worked in the SAME DIRECTION
Not in opposite directions as expected due to antiparallel nature of DNA double strand
➢ 2 forks form from single point of origin
➢ Eukaryote DNA requires MANY POINTS OF REPLICATION of replication due to LARGE DNA
➢ SHORTER Okazaki fragments in eukaryotes
Polymerase δ = main synthesis of lagging strand / has proofreading activity
Polymase ε = elongates LEADING strand
¾ detects an incorrect base due to its geometric shape being different and therefore not fitting the active site of polymerase
Conformational changes occur when correct dNTP pairs with template
= Polymerase closes its open structure around base pair to catalyse phosphodiester bond formation
= Incorrect base pairing show slow confirmational change occurs
Incorrect nucleotide is removed by hydrolysis of phosphodiester bond and then replaced with correct template-primer complex before continuing with DNA synthesis
1. Distortions in DNA helix are recognized
2. NUCLEASES cut nucleotide chain above and below damage
3. DNA HELICASE removes this segment
4. Gap is filled in by DNA polymerase III and Ligase
3 different types of excision repair have been characterized :
2. Low error rate sufficient enough to create variation vital for molecular evolution
Once primer is added, DNA polymerase III elongates strand by added DNA nucleotides to 3'-OH end of growing polymer
LAGGING STRAND = replication occurs AWAY from replication fork
→ Discontinuous elongation by series of shorter double stranded DNA polymers each initiated with own RNA primer (Okazaki fragments)
61 total possible combinations with 3 stop codons
= 2 H bonds in A-T pair, 3 H-bonds in G-C pair
0.34 nm convert to cm = 0.34 x 10⁻⁷
2. 2 thymines stacked on top of eachother bond to form THYMINE DIMERS
Ionising radiation: strand breaks
Nitrous acid: cytosine to uracil, CG → TA
(C is converted to U, U is paired with A, which is then paired with T at the next round of replication
= Overall effect in each case is replacement of a GC pair by an AT pair)
Alkylating agents: guanine modification, GC → AT
Free radicals: strand breaks and base modification
Bulky chemicals: distort DNA helix
1. DNA is wrapped around histones to form nucleosomes
[ DNA is negatively charged (due to phosphate groups) is wrapped tightly around histone core ]
2. Nucleosome is linked to the next one with the help of a linker DNA - further compacted
3. Nucleosomes and linker DNA between are coiled into chromatin fiber → further shortens chromosome
The bacteria were then transferred to a medium containing 14N so that all subsequent DNA chains would be "light"
After 1 generation - all the DNA chains will be identical and contain 1 "heavy" DNA chain from original 15N-labelled double-stranded DNA and 1 new "light" DNA chain
∴ All DNA will be a hybrid of 15N and 14N chains
After 2 generations half the DNA will be exclusively 14N-labelled (the 14N-labelled chains synthesised after 1 generation act as template for the synthesis of a complementary 14N-labelled chain), the original 15N-labelled chains will again act as templates for synthesis of complementary 14N-labelled chains
∴ DNA will be 50:50 mixture of 15N/14N DNA and 14N/14N DNA
After 3 generations once again the 15N-labelled DNA chains will form 15N/14N hybrids and the remaining DNA chains will only comprise "light" chains. 25% of the DNA will now be 15N/14N hybrids and 75% of the DNA 14N/14N
14.4 DNA Replication in Prokaryotes
By the end of this section, you will be able to do the following:
- Explain the process of DNA replication in prokaryotes
- Discuss the role of different enzymes and proteins in supporting this process
DNA replication has been well studied in prokaryotes primarily because of the small size of the genome and because of the large variety of mutants that are available. E. 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 site along the chromosome and proceeding around the circle in both directions. This means that approximately 1000 nucleotides are added per second. Thus, the process is quite rapid and occurs without many mistakes.
DNA replication employs a large number of structural proteins and enzymes, each of which plays a critical role during the process. One of the key players is the enzyme DNA polymerase, also known as DNA pol, which adds nucleotides one-by-one to the growing DNA chain that is complementary to the template strand. The addition of nucleotides requires energy this energy is obtained from the nucleoside triphosphates ATP, GTP, TTP and CTP. Like ATP, the other NTPs (nucleoside triphosphates) are high-energy molecules that can serve both as the source of DNA nucleotides and the source of energy to drive the polymerization. When the bond between the phosphates is “broken,” the energy released is used to form the phosphodiester bond between the incoming nucleotide and the growing chain. In prokaryotes, three main types of polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis DNA pol I is an important accessory enzyme in DNA replication, and along with DNA pol II, is primarily required for repair.
How does the replication machinery know where to begin? It turns out that there are specific nucleotide sequences called origins of replication where replication begins. In E. coli, which has a single origin of replication on its one chromosome (as do most prokaryotes), this origin of replication is approximately 245 base pairs long and is rich in AT sequences. The origin of replication is recognized by certain proteins that bind to this site. An enzyme called helicase unwinds the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication and these get extended bi-directionally as replication proceeds. Single-strand binding proteins coat the single strands of DNA near the replication fork to prevent the single-stranded DNA from winding back into a double helix.
DNA polymerase has two important restrictions: it is able to add nucleotides only in the 5' to 3' direction (a new DNA strand can be only extended in this direction). It also requires a free 3'-OH group to which it can add nucleotides by forming a phosphodiester bond between the 3'-OH end and the 5' phosphate of the next nucleotide. This essentially means that it cannot add nucleotides if a free 3'-OH group is not available. Then how does it add the first nucleotide? The problem is solved with the help of a primer that provides the free 3'-OH end. Another enzyme, RNA primase , synthesizes an RNA segment that is about five to ten nucleotides long and complementary to the template DNA. Because this sequence primes the DNA synthesis, it is appropriately called the primer . DNA polymerase can now extend this RNA primer, adding nucleotides one-by-one that are complementary to the template strand (Figure 14.14).
Question: You isolate a cell strain in which the joining 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 replication fork moves at the rate of 1000 nucleotides per second. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork as the DNA is opening up it does so by causing temporary nicks in the DNA helix and then resealing it. Because DNA polymerase can only extend in the 5' to 3' direction, and because the DNA double helix is antiparallel, there is a slight problem at the replication fork. The two template DNA strands have opposing orientations: one strand is in the 5' to 3' direction and the other is oriented in the 3' to 5' direction. Only one new DNA strand, the one that is complementary to the 3' to 5' parental DNA strand, can be synthesized continuously towards the replication fork. This continuously synthesized strand is known as the leading strand . The other strand, complementary to the 5' to 3' parental DNA, is extended away from the replication fork, in small fragments known as Okazaki fragments , each requiring a primer to start the synthesis. New primer segments are laid down in the direction of the replication fork, but each pointing away from it. (Okazaki fragments are named after the Japanese scientist who first discovered them. The strand with the Okazaki fragments is known as the lagging strand .)
The leading strand can be extended from a single primer, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3' to 5', and that of the leading strand 5' to 3'. A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place. As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA pol I, which uses DNA behind the RNA as its own primer and fills in the gaps left by removal of the RNA nucleotides by the addition of DNA nucleotides. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase , which catalyzes the formation of phosphodiester linkages between the 3'-OH end of one nucleotide and the 5' phosphate end of the other fragment.
Once the chromosome has been completely replicated, the two DNA copies move into two different cells during cell division.
The process of DNA replication can be summarized as follows:
- DNA unwinds at the origin of replication.
- Helicase opens up the DNA-forming replication forks these are extended bidirectionally.
- Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.
- Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
- Primase synthesizes RNA primers complementary to the DNA strand.
- DNA polymerase III starts adding nucleotides to the 3'-OH end of the primer.
- Elongation of both the lagging and the leading strand continues.
- RNA primers are removed by exonuclease activity.
- Gaps are filled by DNA pol I by adding dNTPs.
- The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.
Table 14.1 summarizes the enzymes involved in prokaryotic DNA replication and the functions of each.
3 Main Enzymes of DNA Replications | Cell Biology
A primase is an enzyme which makes the RNA primers required for initiation of Okazaki pieces on the lagging strand. Primase activity needs the formation of a complex of primase and at least six other proteins. This complex is called the primo-some.
The primo-some contains pre-priming proteins—arbitrarily called proteins i, n, n’ and n”—as well as the product of genes dna B and dna C. The primo-some carries out the initial priming activity for leading strand wherein the synthesis takes place continuously in the overall 5′ to 3′ direction.
It also carries out the repeating priming of the synthesis of Okazaki fragments for the lagging strand where the synthesis occurs discontinuously in the overall 3′ to 5′ direction.
The primase shows a very strong preference to initiate with adenosine followed by guanosine and this suggests that initiation of Okazaki fragments may occur at particular sites on the lagging strand. However, the small phage P4, which needs only about 20 Okazaki fragments per round of replication, shows no preferential initiation sites.
The primase that is tightly as­sociated with the eukaryotic DNA polymerase Q is made of two sub-units and shows no stringent sequence requirements. But it does not act at random.
Enzyme # 2. DNA Polymerase:
DNA polymerase is an enzyme that makes a new DNA on a template strand. Both prokaryotic and eukaryotic cells contain more than one species of DNA polymerase enzymes. Only some of these enzymes actually carry out replication and sometimes they are designated as DNA replicases. The others are involved in subsidiary roles in replication and/or par­ticipate repair synthesis of DNA to replace damaged sequences.
DNA polymerase catalyses the formation of a phosphodiester bond between the 3′ hydroxyl group at the growing end of a DNA chain (the primer) and the 5′ phosphate group of the incoming deoxyribonucleoside triphosphate (Fig. 20.8).
Growth is in the 5’→3′ direc­tion and the order in which the deoxyribonucleotides are added is dictated by base pairing to a template DNA chain. Thus, besides four types of deoxyribonucleotides and Mg++ ions, the enzyme requires both primer and template DNA (Figs. 20.9 and 20.10). No DNA polymerase has been found which is able to initiate DNA chains.
DNA polymerases isolated from prokaryotes and eukaryotes differ from each other in several aspects a brief account of these enzyme is given below:
(i) Prokaryotic DNA Polymerase:
There are three different types of prokaryotic DNA polymerases which are called DNA poly­merase I, II and III. These enzymes have been isolated from prokaryotes. DNA polymerase I or Romberg enzyme was first to be isolated from E.coli by Arthur Kornberg etal and was used for DNA synthesis in 1956. Kornberg received (jointly with Severo Ochoa) the Nobel Prize for this work in 1959.
DNA polymerase is a protein of Mr109, 000 in the form of a single polypeptide chain. It contains only one sulphydryl group and one disulphide’ group—the residue at the N- terminus is methionine.
Most of the prokaryotic DNA polymerase I exhibits the following activates:
ii. 3′ → 5′ exonuclease activity.
iii. 5′ → 3′ exonuclease activity.
iv. Excision of the RNA primers used in the initiation DNA synthesis.
DNA polymerase I is mainly responsible for the synthesis of new strand of DNA. This is the polymerase activity. The direction of synthesis of the new strand’ is always 5′ → 3′. But it is estimated that DNA polymerase incorporates wrong bases during DNA replication with a frequency of 10-5. This is not desirable.
Hence DNA polymerase has also 3′ 5′ exonuclease activity (Fig. 20.11) which enables it to proof­read or edit the newly synthesised DNA strand and, thereby, correct the errors made during DNA replication. An exonuclease is an enzyme that degrades nucleic acids from the free ends.
Therefore, whenever the DNA chain being syn­thesised has a terminal mismatch, i.e., insertion of a wrong base in the new chain, the 3’→ 5′ exonuclease activity of DNA polymerase I in reverse direction clips off the wrong base and immediately the same enzyme, i.e., DNA polymerase I, reinitiates the synthesis of correct base in the growing new chain.
Therefore, due to this dual activity of DNA polymerase I, the chance of errors in DNA replication is reduced.
The 5′ → 3′ exonuclease activity of DNA polymerase I is also very important. It func­tions in the removal of the DNA segment damaged by the irradiation of ultraviolet ray and other agents. An endonuclease (degrades nucleic acid by making internal cut) must cleave the DNA strand close to the site of damage before 5′ → 3′ exonuclease action of the DNA polymerase I may take place.
The 5′ → 3′ exonuclease activity of DNA polymerase I also functions in the removal of RNA primers from DNA. The ribonucleotides are Immediately replaced by deoxyribonucleotides due to the 5′ → 3′ polymerase activity of the enzyme.
The prokaryotic DNA polymerase II was discovered in pol A – mutant of E.coli. Pol A is a gene responsible for the synthesis of polymerase I. Therefore, the mutant of pol A – are deficient in DNA polymerase I or Kornberg enzyme. But, in absence of DNA polymerase I, replication of DNA also takes place in such mutant type.
Therefore, it is obvious that DNA poly­merase II plays a role in DNA replication of such mutant. DNA polymerase II has 5’→ 3′ polymerase activity but it uses gapped DNA template. This enzyme also has the 3′ → 5′ but not the 5′ → 3′ exonuclease activity. The function of E.coli DNA polymerase II in vivo is unknown.
Prokaryotic DNA polymerase III was also discovered in pol A – mutant. There is a strong evidence that unlike DNA polymerase I and II, polymerase III is essential for DNA synthe­sis. The best template for DNA polymerase III is double-stranded DNA with very small gaps containing 3′-OH priming ends. In the DNA polymerase II, the core enzyme is tightly associated with two small sub-units.
The core enzyme has both 3′ → 5′ exonuclease (which could be involved in proof-reading) and 5’→ 3′ exonuclease activities, although the latter is only manifest in vitro on duplex DNA with a single-stranded 5′ tail.
This enzyme has a higher affinity for nucleotide triphosphate than DNA polymerase I and II and catalyses the synthesis of DNA chains at very high rates, i.e., 10-15 times the rate of polymerase I. The major properties of the three DNA polymerases are summarised in Table 22.3.
A DNA polymerase molecule has four func­tional sites which are involved in polymerase activity.
These sites are:
(iii) Primer terminus site, and
The template site binds to the DNA strand functioning as template during DNA replica­tion and holds it in the correct orientation. The primer site is the site where the primer chains to which the nucleotides will be added are attached.
The primer terminus site ensures that the primer binding to the primer site has a free 3′- OH. A primer without a free 3′-OH is not able to bind to this site.
The triphosphate site is the site for bind­ing deoxyribonucleotide 5′-triphosphate that is complementary to the corresponding nucleotide of the template and catalyses the formation of phosphodiester bond between the 5′ phosphate of this nucleotide and the 3′-OH of the terminal primer nucleotide. In addition, there is a 3’→ 5′ exonuclease site and a 5′-3′ exonuclease site of DNA poly­merase I.
(ii) Eukaryotic DNA Polymerase:
In higher eukaryotes, there are at least four DNA polymerases known as α, β,y and δ and a fifth (ɛ) has recently been described. In yeast DNA, polymerase I corresponds to DNA polymerase a, polymerase II to e, polymerase III to 6 and polymerase m to S and they have renamed accordingly.
Polymerase α is present in the nuclei of the cell. DNA polymerase a shows optimal activity with a gapped DNA template but shows a remarkable ability to use single-stranded DNA by forming transient hairpins. It will not bind to duplex DNA.
The native, undegraded enzyme consists of a 180 K Da polymerase together with three sub- units—the 60 and 50 K Da sub-units of about 70, 60 and 50 K Da. Association of the 180 K Da polymerase with the 70KDa protein makes the 3’→ 5′ exonuclease activity of the larger sub-units comprise a primase activity which allows the enzyme to initiate replication on unprimed single-stranded cyclic DNAs.
There­fore, polymerase a have dual activity, i.e., both the polymerase and primase activity. The association of primase with DNA polymerase α is restricted to the DNA synthetic phase.
Polymerase β is also present in the nuclei. It shows optimal activity with native DNA acti­vated by limited treatment with native DNA-ase I to make single-stranded nicks and short gaps bearing 3′-OH priming termini and also shows negligible activity with denatured DNA. DNA polymerase β is believed to play a role in repair of DNA.
Polymerase δ is present in the dividing cell and have got similar properties polymerase a, but having 3′ → 5′ exonuclease activity. The activity of polymerase δ is dependent on activity on two auxiliary proteins: cyclin and activator I.
Due to presence of approximately equal activities of DNA polymerase α and δ it has been proposed that they act as a dimer at the replication fork with the highly processive polymerase δ acting on the leading strand and the primease-associated polymerase a acting on the lagging strand.
Cyclin or PCNA (proliferating cell nuclear antigen) independent form of DNA polymerase 6 is known as polymerase e which has two ac­tive polymerase sub-units of 220 and 145 K Da. DNA polymerase e is also probably involved in replication and it has been proposed that it takes over from DNA polymerase a in the synthesis of Okazaki fragments.
Polymerase y is found in small amount in animal cells. It is also found in mitochon­dria and chloroplasts and is believed to be responsible for replication of the chromosome of these organelles. DNA polymerase 7 isolated from chick embryos is a tetramer having four identical sub-units. It has also a proof-reading exonuclease activity.
Enzyme # 3. DNA Ligases:
DNA ligase is an important enzyme involved in DNA replication. DNA ligases catalyse the formation of a phosphodiester bond between the free 5′ phosphate end of an oligo or polynu­cleotide and the 3′-OH group of a second oligo or polynucleotide next to it.
A ligase-AMP complex seems to be an obligatory intermediate and is formed by reaction with NAD in case of E.coli and B. subtilis and with ATP in mam­malian and phage-infected cells.
The adenyl group is then transferred from the enzyme to the 5′ phosphoryl terminus of the DNA. The activated phosphoryl group is then attached by the 3′-hydroxyl terminus of the DNA to form a phosphodiester bond. DNA ligases join successive Okazaki fragments produced during discontinuous DNA replication and seal the nicks left behind by DNA polymerase.
The enzymes so far discussed are required for the synthesis of DNA on parental tem­plate strand of DNA. But in certain RNA virus or retrovirus, there is an enzyme—called RNA-dependent DNA polymerase or reverse transcriptase—which uses parental RNA strand as a template for the synthesis of DNA.
The immediate product of this enzyme activity is the formation of double-stranded RNA-DNA hybrid which is the result of the synthesis of a complementary strand of DNA using single- stranded viral RNA as template. This enzyme uses viral RNA as template.