1. DNA Replication

Genetica per Scienze Naturalia.a. 05-06 prof S. Presciuttini

1. DNA Replication In both prokaryotes and eukaryotes, DNA replication In both prokaryotes and eukaryotes, DNA replication

occurs as a prelude to cell division. This DNA replication occurs as a prelude to cell division. This DNA replication phase is called the phase is called the S (synthesis) phaseS (synthesis) phase. The two daughter . The two daughter DNA molecules formed from replication eventually DNA molecules formed from replication eventually become chromosomes in their own right in the daughter become chromosomes in their own right in the daughter cells.cells.

As with all phenomena that involve nucleic acids, the As with all phenomena that involve nucleic acids, the basic machinery of DNA replication depends on basic machinery of DNA replication depends on complementaritycomplementarity of DNA molecules and on the ability of of DNA molecules and on the ability of proteins to form specific interactions with DNA of specific proteins to form specific interactions with DNA of specific sequences.sequences.


2. The model of Watson and Crick

The model of DNA replication proposed by Watson and The model of DNA replication proposed by Watson and Crick is based on the hydrogen-bonded specificity of the Crick is based on the hydrogen-bonded specificity of the base pairs. Complementary strands are shown in different base pairs. Complementary strands are shown in different colors. The fact that colors. The fact that new strands can grow only in the new strands can grow only in the 55’’-to-3-to-3’’ direction direction adds complexities to the detailed adds complexities to the detailed mechanism of replicationmechanism of replication..

If this model is correct, then each daughter molecule If this model is correct, then each daughter molecule should contain one parental nucleotide chain and one should contain one parental nucleotide chain and one newly synthesized nucleotide chain. This prediction has newly synthesized nucleotide chain. This prediction has been tested in both prokaryotes and eukaryotes. A little been tested in both prokaryotes and eukaryotes. A little thought shows that there are at least three different ways thought shows that there are at least three different ways in which a parental DNA molecule might be related to the in which a parental DNA molecule might be related to the daughter molecules. These hypothetical modes are called daughter molecules. These hypothetical modes are called semiconservative (the Watson-Crick model), conservative, semiconservative (the Watson-Crick model), conservative, and dispersiveand dispersive


3. Three alternative patterns for DNA replicationIn semiconservative replication, In semiconservative replication, each daughter duplex contains one each daughter duplex contains one parental and one newly synthesized parental and one newly synthesized strand. However, in conservative strand. However, in conservative replication, one daughter duplex replication, one daughter duplex consists of two newly synthesized consists of two newly synthesized strands, and the parent duplex is strands, and the parent duplex is conserved. Dispersive replication conserved. Dispersive replication results in daughter duplexes that results in daughter duplexes that consist of strands containing only consist of strands containing only segments of parental DNA and segments of parental DNA and newly synthesized DNAnewly synthesized DNA


4. The Meselson-Stahl experiment In 1958, Matthew Meselson and Franklin Stahl set out to distinguish among theIn 1958, Matthew Meselson and Franklin Stahl set out to distinguish among the

three modelsthree models. They grew E. coli cells in a medium containing the . They grew E. coli cells in a medium containing the heavy isotope of heavy isotope of nitrogen nitrogen 1515NN rather than the normal light ( rather than the normal light (1414N) form. This isotope was inserted into N) form. This isotope was inserted into the nitrogen bases, which then were incorporated into newly synthesized DNA the nitrogen bases, which then were incorporated into newly synthesized DNA strands.strands.

After many cell divisions in After many cell divisions in 1515N, the N, the DNA of the cells were well labeled DNA of the cells were well labeled with the heavy isotope. The cells were with the heavy isotope. The cells were then removed from the then removed from the 1515N medium N medium and put into a and put into a 1414N medium; after N medium; after one one and two cell divisionsand two cell divisions, samples were , samples were taken. DNA was extracted from the taken. DNA was extracted from the cells in each of these samples and put cells in each of these samples and put into a solution of cesium chloride into a solution of cesium chloride (CsCl) in an ultracentrifuge(CsCl) in an ultracentrifuge..


5. Centrifugation of DNAin a cesium chloride (CsCl) gradient

If cesium chloride is spun in a centrifuge at tremendously high speeds (50,000 rpm) If cesium chloride is spun in a centrifuge at tremendously high speeds (50,000 rpm) for many hours, the cesium and chloride ions tend to be pushed by centrifugal force for many hours, the cesium and chloride ions tend to be pushed by centrifugal force toward the bottom of the tube.toward the bottom of the tube. Ultimately, Ultimately, a gradient of Cs+ and Cl ionsa gradient of Cs+ and Cl ions is is established in the tube, with the highest ion concentration at the bottom.established in the tube, with the highest ion concentration at the bottom.

Molecules of DNA in the solution also are pushed toward the bottom by centrifugal Molecules of DNA in the solution also are pushed toward the bottom by centrifugal force. But, as they travel down the tube, they encounter the increasing salt force. But, as they travel down the tube, they encounter the increasing salt concentration, which tends to push them back up owing to the buoyancy of DNA concentration, which tends to push them back up owing to the buoyancy of DNA (its tendency to float). Thus, the DNA finally "settles" at some point in the tube (its tendency to float). Thus, the DNA finally "settles" at some point in the tube where where the centrifugal forces just balance the buoyancythe centrifugal forces just balance the buoyancy of the molecules in the of the molecules in the cesium chloride gradient.cesium chloride gradient.

The buoyancy of DNA depends on its density (which in turn depends on the ratio of The buoyancy of DNA depends on its density (which in turn depends on the ratio of GC to AT base pairs). The presence of the heavier isotope of nitrogen changes the GC to AT base pairs). The presence of the heavier isotope of nitrogen changes the buoyant density of DNA. The DNA extracted from cells grown for several buoyant density of DNA. The DNA extracted from cells grown for several generations on generations on 1515N medium can be readily distinguished from the DNA of cells N medium can be readily distinguished from the DNA of cells grown on grown on 1414N medium by the N medium by the equilibrium position reached in a cesium chloride equilibrium position reached in a cesium chloride gradientgradient. Such samples are commonly called heavy and light DNA, respectively.. Such samples are commonly called heavy and light DNA, respectively.


6. The proof of the semiconservative modelMeselson and Stahl found that, Meselson and Stahl found that, one generation one generation afterafter the heavy cells were moved to the heavy cells were moved to 1414N N medium, the DNA formed a single band of an medium, the DNA formed a single band of an intermediate density between the densities of the intermediate density between the densities of the heavy and light controls. After two generations heavy and light controls. After two generations in in 1414N medium, the DNA formed two bands: one N medium, the DNA formed two bands: one at the intermediate position, the other at the light at the intermediate position, the other at the light positionposition..

This result would be expected from the This result would be expected from the semiconservative mode of replication; in fact, semiconservative mode of replication; in fact, the result is compatible with only this mode if the result is compatible with only this mode if the experiment begins with chromosomes the experiment begins with chromosomes composed of individual double helicescomposed of individual double helices


7. Harlequin chromosomes With the use of a more modern staining technique, it is now possible to visualize the With the use of a more modern staining technique, it is now possible to visualize the

semiconservative replication of chromosomes at semiconservative replication of chromosomes at mitosismitosis. . In this procedure, the In this procedure, the chromosomes go through two rounds of replication in the presence of chromosomes go through two rounds of replication in the presence of bromodeoxyuridine (BUdR),bromodeoxyuridine (BUdR), which replaces which replaces thymidinethymidine in the newly synthesized in the newly synthesized DNA. The chromosomes are then stained with Giemsa stain, producing the DNA. The chromosomes are then stained with Giemsa stain, producing the appearance shown. (The light blue lines represent the BUdR-substituted strands.)appearance shown. (The light blue lines represent the BUdR-substituted strands.)


8. Visualizing sister chromatids

If cells dividing in culture are treated with If cells dividing in culture are treated with BrdU during S phase, the cells are fooled BrdU during S phase, the cells are fooled into incorporating it — instead of into incorporating it — instead of thymidine — into their DNA. thymidine — into their DNA. One of the properties of the resulting One of the properties of the resulting DNA is that it fails to take up stain in a DNA is that it fails to take up stain in a normal way. normal way. When cells are allowed to duplicate their When cells are allowed to duplicate their chromosomes once in BrdU, the chromosomes once in BrdU, the chromosome that appear at the next chromosome that appear at the next metaphase stain normally. metaphase stain normally. However, when the cells duplicate their However, when the cells duplicate their chromosomes a second time in BrdU, one chromosomes a second time in BrdU, one of the of the sister chromatidssister chromatids that appears at that appears at the next metaphase stains normally, while the next metaphase stains normally, while its sister chromatid does not.its sister chromatid does not.


9. DNA polymerases In the late 1950s, Arthur In the late 1950s, Arthur

Kornberg successfully Kornberg successfully identified and purified the first identified and purified the first DNA polymerase, an enzyme DNA polymerase, an enzyme that catalyzes the replication that catalyzes the replication reaction.reaction.

This reaction works only with This reaction works only with the triphosphate forms of the the triphosphate forms of the nucleotides (such as nucleotides (such as deoxyadenosine triphosphate, deoxyadenosine triphosphate,

or dATP).or dATP).


10. DNA polymerases in E. coli We now know that there are three DNA polymerases in E. coli. The We now know that there are three DNA polymerases in E. coli. The

first enzyme that Kornberg purified is called DNA polymerase I or first enzyme that Kornberg purified is called DNA polymerase I or pol Ipol I. This enzyme has three activities, which appear to be located in . This enzyme has three activities, which appear to be located in different parts of the molecule:different parts of the molecule: 1. a 1. a polymerasepolymerase activity, which catalyzes chain growth in the 5 activity, which catalyzes chain growth in the 5’’ 3 3’’ direction; direction; 2. a 32. a 3’’ 5 5’’ exonucleaseexonuclease activity, which removes mismatched bases; and activity, which removes mismatched bases; and 3. a 53. a 5’’ 3 3’’ exonucleaseexonuclease activity, which degrades double-stranded DNA. activity, which degrades double-stranded DNA.

Subsequently, two additional polymerases, Subsequently, two additional polymerases, pol IIpol II and and pol IIIpol III, were , were identified in E. coli. Pol II may repair damaged DNA. Pol III, together identified in E. coli. Pol II may repair damaged DNA. Pol III, together with pol I, has a role in the replication of E. coli DNAwith pol I, has a role in the replication of E. coli DNA


11. DNA replication fork The complete complex, or holoenzyme, of pol III contains The complete complex, or holoenzyme, of pol III contains at least 20 different at least 20 different polypeptide subunitspolypeptide subunits, although the catalytic "core" consists of only three subunits. , although the catalytic "core" consists of only three subunits. The pol III complex will complete the replication of single-stranded DNA if there is The pol III complex will complete the replication of single-stranded DNA if there is at least a short segment of duplexat least a short segment of duplex already present. already present.


12. Prokaryotic origins of replication E. coli replication begins from a fixed originE. coli replication begins from a fixed origin,, termed oriC termed oriC,, but then proceeds but then proceeds

bidirectionally (with moving forks at both ends of the replicating piece). It is bidirectionally (with moving forks at both ends of the replicating piece). It is 245 bp245 bp long and has several components. First, there is a side-by-side, or tandem, set of 13-long and has several components. First, there is a side-by-side, or tandem, set of 13-bp sequences, bp sequences, which are nearly identicalwhich are nearly identical. There is also a set of binding sites for a . There is also a set of binding sites for a protein, the protein, the DnaA proteinDnaA protein. An initial step in DNA synthesis is the unwinding of the . An initial step in DNA synthesis is the unwinding of the DNA at the origin in response to binding of the DnaA protein.DNA at the origin in response to binding of the DnaA protein.


13. A replicating E. coli chromosomeThe DNA has been labeled with 3H-deoxythymidine, and the radioactivity has been detected by overlaying the replicating chromosome with photographic emulsion. The autoradiograph shows that the E. coli chromosome has two replication forks.

Although there seem to be two bubbles of replication, actually the point where the two smaller bubbles meet is actually just where two strands of DNA are laying across one another


14. Eukaryotic origins of replication Bacteria such as E. coli usually require a 40-minute replication-Bacteria such as E. coli usually require a 40-minute replication-

division cycle, but, in eukaryotes, the cycle can vary from 1.4 division cycle, but, in eukaryotes, the cycle can vary from 1.4 hours in yeast to 24 hours in cultured animal cells and may last hours in yeast to 24 hours in cultured animal cells and may last from 100 to 200 hours in some cells.from 100 to 200 hours in some cells.

Eukaryotes have to solve the problem of coordinating the Eukaryotes have to solve the problem of coordinating the replication of more than one chromosome, as well as replicating replication of more than one chromosome, as well as replicating the complex structure of the chromosome itself.the complex structure of the chromosome itself.

In eukaryotes, replication proceeds from multiple points of In eukaryotes, replication proceeds from multiple points of origin.origin.

EExperiments in yeast indicate the existence of xperiments in yeast indicate the existence of aboutabout 400 400 replication origins distributed among the 17 chromosomes, and replication origins distributed among the 17 chromosomes, and in humans there are estimated to be more than 10,000 growing in humans there are estimated to be more than 10,000 growing forksforks


15. Replication bubbles in the fruit fly

At least 20 different bubbles, therefore with at least 40 different replication forks, can be observed in this electron micrograph (and accompanying drawn representation of the electron micrograph.) The large number of replication origins in eukaryotic chromosomes vs. E. coli's one, enables the slower replication apparatus to copy the larger eukaryotic genome in approximately the same amount of time as the prokaryotic genome is replicated

Electron micrograph of replicating DNA in the embryo of the fruit fly D. melanogaster


16. Replication bubbles

Electron micrograph of Electron micrograph of DNA extracted from DNA extracted from rapidly dividing nuclei rapidly dividing nuclei of early D. Melanogaster of early D. Melanogaster embryos. The arrows embryos. The arrows mark replication mark replication bubbles; the diameters of bubbles; the diameters of DNA chain in both arms DNA chain in both arms of these bubbles indicate of these bubbles indicate that they are double-that they are double-stranded.stranded.


17. Priming DNA synthesisDNA polymerases can extend a chain but DNA polymerases can extend a chain but cannot cannot start a chainstart a chain. Therefore, DNA synthesis must first . Therefore, DNA synthesis must first be initiated with a be initiated with a primerprimer, or short oligonucleotide, , or short oligonucleotide, that generates a segment of that generates a segment of duplex DNAduplex DNA..

RNA primersRNA primers are synthesized either by RNA are synthesized either by RNA polymerase or by an enzyme termed polymerase or by an enzyme termed primaseprimase. . Primase synthesizes a short (approximately 30 bp Primase synthesizes a short (approximately 30 bp long) stretch of RNA complementary to a specific long) stretch of RNA complementary to a specific region of the chromosome.region of the chromosome.

The The RNA chain is then extended with DNARNA chain is then extended with DNA by by DNA polymerase. DNA polymerase. E. coliE. coli primase forms a complex primase forms a complex with the template DNA, and additional proteins, such with the template DNA, and additional proteins, such as DnaB, DnaT, Pri A, Pri B, and Pri C. The entire as DnaB, DnaT, Pri A, Pri B, and Pri C. The entire complex is termed a complex is termed a primosomeprimosome..


18. Leading strand and lagging strandDNA polymerases synthesize new chains only in DNA polymerases synthesize new chains only in the the 55’’ 33’’ direction and therefore, because of the direction and therefore, because of the antiparallel nature of the DNA molecule, move in a antiparallel nature of the DNA molecule, move in a 33’’ 55’’ direction on the template strand. The direction on the template strand. The consequence of this polarity is that while one new consequence of this polarity is that while one new strand, the leading strand, is synthesized strand, the leading strand, is synthesized continuously, the other, the lagging strand, must be continuously, the other, the lagging strand, must be synthesized in synthesized in short, discontinuous segmentsshort, discontinuous segments. . The The addition of nucleotides along the template for the addition of nucleotides along the template for the lagging strandlagging strand must proceed toward the template's must proceed toward the template's 55’’ end (because replication always moves along the end (because replication always moves along the

template in a 3template in a 3’’ 55’’ direction direction so that the new so that the new strand can grow 5strand can grow 5’’ 33’’). Thus, the new strand ). Thus, the new strand must grow in a must grow in a direction opposite that of the direction opposite that of the movement of the replication forkmovement of the replication fork..


19. Discontinuous synthesisAs fork movement exposes a new section of lagging-strand template, a new lagging-As fork movement exposes a new section of lagging-strand template, a new lagging-strand fragment is begun and proceeds away from the fork until it is stopped by the strand fragment is begun and proceeds away from the fork until it is stopped by the preceding fragment.preceding fragment.

In E. coli, pol III carries out most of the DNA synthesis on both strands, and In E. coli, pol III carries out most of the DNA synthesis on both strands, and pol I fills pol I fills in the gapsin the gaps left in the lagging strand, which are then sealed by the enzyme DNA left in the lagging strand, which are then sealed by the enzyme DNA ligaseligase..

DNA ligases join broken pieces of DNA by catalyzing the formation of a DNA ligases join broken pieces of DNA by catalyzing the formation of a phosphodiester bond between the 5phosphodiester bond between the 5’’ phosphate end of a hydrogen-bonded nucleotide phosphate end of a hydrogen-bonded nucleotide and an adjacent 3and an adjacent 3’’ OH group OH group. . It is the only enzyme that It is the only enzyme that can seal DNA chainscan seal DNA chains..


20. Steps in DNA synthesisa) a) The primers for the The primers for the

discontinuous synthesis on the discontinuous synthesis on the lagging strand are synthesized lagging strand are synthesized by by primaseprimase..

b) b) The primers are extended by The primers are extended by DNA polymerase to DNA polymerase to yield DNA yield DNA fragmentsfragments that were first that were first detected by Reiji Okazaki and detected by Reiji Okazaki and are termed are termed Okazaki Okazaki fragmentsfragments..

c)c) The 5 The 5’’ 3 3’’ exonucleaseexonuclease activity of pol I activity of pol I removes the removes the primersprimers and fills in the gaps and fills in the gaps with DNA, with DNA,

d) d) which are which are sealedsealed by DNA by DNA ligaseligase. .


21. A comprehensive view of the replication fork


22. Other DNA-modifying enzymes HelicasesHelicases are enzymes that are enzymes that disrupt the hydrogen bondsdisrupt the hydrogen bonds that hold the two DNA that hold the two DNA

strands together in a double helix. Among strands together in a double helix. Among E. coliE. coli helicases are the DnaB protein helicases are the DnaB protein and the Rep protein. The Rep protein may help to unwind the double helix ahead of and the Rep protein. The Rep protein may help to unwind the double helix ahead of the polymerase. The unwound DNA is stabilized by the single-stranded binding the polymerase. The unwound DNA is stabilized by the single-stranded binding (SSB) protein, which binds to the single-stranded DNA and retards reformation of (SSB) protein, which binds to the single-stranded DNA and retards reformation of the duplex.the duplex.

The action of helicases during DNA replication generates The action of helicases during DNA replication generates twiststwists in the circular in the circular DNA that need to be removed to allow replication to continue. Circular DNA can be DNA that need to be removed to allow replication to continue. Circular DNA can be twisted and coiledtwisted and coiled, much like the extra coils that can be introduced into a rubber , much like the extra coils that can be introduced into a rubber band.band.

This This supercoilingsupercoiling can be created or relaxed by enzymes termed can be created or relaxed by enzymes termed topoisomerasestopoisomerases. . There are two basic types of isomerases. Type I enzymes induce a There are two basic types of isomerases. Type I enzymes induce a single-stranded single-stranded breakbreak into the DNA duplex. Type II enzymes cause a into the DNA duplex. Type II enzymes cause a break in both strandsbreak in both strands. In . In E. E. colicoli, topo I and topo III are examples of type I enzymes, whereas , topo I and topo III are examples of type I enzymes, whereas gyrasegyrase is an is an example of a type II enzyme.example of a type II enzyme.


23. The action of topoisomerases Untwisting of the DNA strands to open the replication fork causes Untwisting of the DNA strands to open the replication fork causes

extra twisting at other regions, and the supercoiling releases the strain extra twisting at other regions, and the supercoiling releases the strain of the extra twisting. During replication, of the extra twisting. During replication, gyrasegyrase is needed to remove is needed to remove positive supercoilspositive supercoils ahead of the replication fork ahead of the replication fork

Swivel function of topoisomerase during replication. Extra-twisted (positively supercoiled) regions accumulate ahead of the fork as the parental strands separate for replication. A topoisomerase is required to remove these regions, acting as a swivel to allow extensive replication.

Documents

1. DNA Replication