Dna Replication

DNA replication

Watson and Crick duplex structure of DNA immediately suggested how genetic material was replicated from one generation to the next. The realization that bacterial genomes and eukaryotic chromosomes consist of single DNA millimeters to centimeters in length raised a series of structural and biochemical questions about DNA replication. Questions to be answered by the course: How does the replication begin; how does it progress along the chromosome? What mechanism ensure that only one round of replication occures before cell division? Which enzymes take part in DNA synthesis, and what are their functions? Importantly – as DNA serves as genetic link between the generations, the base sequence must not only be copied correctly during replication, but also maintained throughout lifespan. To keep DNA sequences accurate cells possess enzymes that catalyze DNA repair. DNA recombination provides a mechanism for generating new genetic diversity.

1

Watson and Crick’s model of replication The base-pairing principal inherent for Watson-Crick model suggests that the two new strands are copied from two old strands. …two chains unwind and separate. Each chain then acts as a template for the formation onto itself of a new companion chain…

Upon the replication of double-helix each of the two daughter molecules will have one old strand (parental) and one newly made strand. Such a process is called semiconservative replication.

Alternative models or replication To explain the phenomenon of heredity, biological information must be accurately copied and transmitted from each cell to all of its progeny. Three ways for DNA molecules to replicate may be considered, each obeying the rules of complementary base pairing. • Conservative replication would leave intact the original DNA molecule and generate a completely new molecule. •Dispersive replication would produce two DNA molecules with sections of both old and new DNA interspersed along each strand. •Semiconservative replication would produce molecules with both old and new DNA, but each molecule would be composed of one old strand and one new one.

2

Three models of DNA replication.

The Meselson-Stahl Experiment Predictions Bacteria in medium with 15N

Conservative

Semiconservative

Dispersive

Centrifuge DNA sample after 20 min Second replication Bacteria transferred to medium Centrifuge with 14N DNA sample after 40 min centrifugation CsCl gradient

3

Summary of Meselson-Stahl experiment The replication in bacteria is semiconservative The conservative model could be ruled out after the first round of replication, since the only one intermediate bend was present Dispersive model was ruled out by two major observations: • when the hybrid molecule was heat denatured after the first round of replication, the density of the single strands corresponded to either 15N- or 14N-profile but not an intermediate; • the second round of replication resulted in the presence of two bends in semiconservative (intermediate and 14N forms) and would result in one shifted bend in dispersive model.

Semidiscontinuous replication Both strands can not replicate continuously – as polymerase goes only 5’ to 3’. Leading strand- continuously; lagging strand – discontinuously. Discontinuity comes from its direction – opposite to the direction of fork moving. Okazaki’s model of semidiscontinuous replication made two predictions: 1. Because at least half of newly synthesized DNA appears in pieces, one ought to be able to label them before them are stitched together allowing short pulces of radioactive DNA precursor. 2. 2. If one eliminates enzyme DNA ligase is responsible for stitching these short pieces ought to be detectable.

4

Experiment: •T4 phage – simple •Shorter and shorter pulses of H3 labeled thymidine (2 sec). •Measured sizes of DNA that was synthesized •Already at 2 seconds DNA was visible in the gradient – short pieces about 1000-2000nt. •Increasing pulse time –labeled DNA appeared much nearer to the bottom of the tube – result of attaching the small, newly formed pieces of labeled DNA to much larger, preformed pieces that were made before the labeling began. They did not show up before ligase joined small labeled pieces to them. •Small pieces of DNA that are initial products of replication are known as Okazaki fragments.

5

Most DNA replication is bidirectional Starts at the origin -defined sequence of base pairs Each region served by one DNA origin is called a replicon

Some linear DNA viruses

Certain plasmids

Most common for eukaryotes and prokaryotes

Bidirectional DNA replication

6

Bidirectional DNA replication in eukaryotes

Mechanisms of the strand growth Mechanism one: one strand derives from the origin and the other strand derives from another origin.Only one strand of the duplex grows at each growing point. In this mechanism, which operates in linear DNA viruses such as adenovirus, the ends of the DNA molecules serve as fixed sites for the initiation and termination of replication. Mechanism two entails one origin –one growing fork (the point where DNA replication occurs), which moves along the DNA in one direction with both strands of DNA being copied. Certain bacterial plasmids replicate in such way.

7

Mechanisms of the strand growth A third mechanism is that synthesis might start at a single origin and proceed both directions , so that both strands are copied at each growing fork. The available evidence suggests that the third alternative is most generally used in prokaryotic and eukaryotic cells – replication proceeds bidirectionally from given starting site with both strands being copied at each fork. In circular DNA molecules present in bacteria, plasmids and some viruses one origin often suffices – two resulting growing forks merge on the opposite site of the circle to complete replication. However, long linear chromosomes of eukaryotes contain multiple origins; origins the two rowing forks from particular origin continue to advance untill they meet advancing forks growing from neighboring origins.

Such studies have revealed clusters of active replicons

Replicating mammalian cells were exposed first to high then to low concentration of H3Thymidine – DENA will be heavily labeled near the origin and lightly later.

8

Most DNA replication is bidirectional Prokaryotic chromosomes have a single origin of replication with two replication forks Much larger eukaryotic chromosomes have many origins of replication Each region served by one DNA origin is called replicon. First evidence of bidirectional fork growth came from fiber autoradiography of labeled DNA molecules from mammalian cultured cells. Such studies revealed clusters of active replicons, each of which contain 2 growing forks moving away from a central origin.

Demonstration of bidirectional chain growth from a single origin in viral DNA

origin

EcoRI restriction site

Circular viral chromosome

Replication bubble

Time of replication

EcoRI

EM - replication bubbles The replication viral DNA from SV40-infected cells was cut by EcoRI, which recognizes single site and examined by electron microscopy. Series of ever large bubbles whose centers maps to the same site. The EM pictures showed a collection of increasingly growing replication bubbles, the centers of which are a constant distance from each end of the cut molecules, thus indicating that chain growth occurs in two directions from a common origin.

9

Number of growing forks and their rate of movement 9In E. coli cells 9it takes 42 minutes to replicate the single circular chromosome that has 4 639 221 bp and is about 4.1mm in length. 9Since the chromosome is duplicated from one origin by two growing forks, we can calculate that the rate of the fork movement is about 1000bp/second/fork.

Number of growing forks and their rate of movement 9The rate of fork movement in human cells, based on fiberlabeling experiments, is only about 100bp/second/fork. 9The entire human genome of 3 x 109 bp replicates in about 8 hours, suggesting that human genome might have about 1000 forks. 9However, fiber autoradiography and electron microscopy indicate that growing forks are spaced closer than 3 x 106 apart. 9A most likely estimate is that human genome contains 10 000100 000 replicons, each of which is actively replicating for only part of the 8 hours required for replication of the entire genome.

10

DNA replication begins at specific chromosomal sites

DNA replication as many other processes is controlled by initiation step. Replication of DNA begins at a defined sequence of base pairs near the center of the replication bubbles, called replication origin. A replication origin is a stretch of DNA that is necessary and sufficient for replication of a circular DNA molecule, usually a plasmid or virus, in an appropriate host cell. In yeast this definition has been refined to include sequences that direct replication once per S phase – the period of the cell cycle when chromosomal duplication takes place.

Replication bubbles

11

DNA Replication Origin

Replication origin = site on the DNA double helix where replication is initiated. •site where the double helix first opens ---> replication bubble. •consist of specific nucleotide sequences recognized by initiator proteins. oA-T rich (easier to separate) o100 bp (base pairs) in length

Number of replication origins •Prokaryotes o1 replication origin per chromosome oreplication rate = 500 nucleotides per sec. •Eukaryotes omultiple replication sites on each chromosome. oreplication rate = 50 nucleotides per sec. In eukaryotes replication origins are activated in clusters of 20 to 80 adjacent origins = replication units. The pattern of replication is controlled, temporally and spatially.

12

DNA Replication Origin of E.coli 9E. coli replication origin oriC is an ≈240bp DNA segment present at the start site for the replication of the E. coli chromosomal DNA. 9Plasmids or any other circular DNAs containing oriC are capable of independent and controlled replication in E. coli cells. 9Comparison of oriC with the origins of five other bacterial species including the distant species Virbio harveyi revealed that all contain repetitive 9bp and AT-rich 13 bp sequences, called 9-mers (dnaA boxes) and 13-mers. 9These are binding sites for DnaA protein that initiates replication. 9In addition, the E. coli genome contains a segment of DNA with a relatively high A+T content adjacent to the oriC. This sequence appears to facilitate local melting of DNA segments onto which the replication machinery is loaded. 9After E. coli replication has initiated, replication origins in the two daughter DNA duplexes become linked to specific proteins on the plasma membrane. As the cell wall divides and forms this linkage assures that one of the daughter DNA duplexes is delivered to each daughter cell.

DNA Replication Origin of E.coli

1

13

17

5’ GATCTNTT TATTT

29

GATCTNTT TATTT CTAGANAAATAAA

3’ CTAGANAAATAAA 58

32

44

GATCTNTT TATTT CTAGANAAATAAA

66

TGTGGATAA ACACCTATT 166

174

TTATACACA AATATGTGT

201

209

TTTGGATAA AAACCTATT

240

248

TTATCCACA AATAGGTGT

3’ 5’

Consensus sequence of the minimal bacterial replication origin based on analyses of genomes from six bacterial species 13 bp repetitive sequences are rich in A and T. The 9bp sequences exist in both orientations. These sequences are referred as 13-mers ans 9-mers.

13

DNA Replication Origin of E.coli Cell wall Plasma membrane origins

The origins of the replicated chromosomes have independent points of attachment to the membrane and thus move further apart as new membrane and cell wall forms midway along the length of the cell.

DNA Replication Origin of E.coli, oriC and comparisons with other origin sequences in other bacteria.

14

Yeast autonomously replicating sequences

¾Each yeast chromosome has multiple origins of replication: about 400 origins exist ¾on 17 chromosomes of S. cerevisiae. ¾Each yeast origin, called autonomously replicating sequence (ARS), confers on a plasmid the ability to replicate in yeast and is a required element for YACs. ¾Detailed mutational analysis of one ≈180 bp ARS called ARS1 revealed only one element, a 15-bp segment, designated element A, stretching from position 114 to 128. ¾Three other short segments – elements B1, B2 and B3 – increase the efficiency of ARS functioning. Comparison of the sequences required for functioning of many different DNA segments that act as ARSs led to recognition of an 11-bp consensus sequence: (5’) A/T-T-T-T-A-T-A/G-T-T-T-A/T (3’)

¾Element A in ARS1 is identical in 10 out of 11 positions of the consensus sequence, and element B2 – in 9 of 11. ¾DNA footprinting revealed that 6 different proteins called the ORC (origin recognition complex) binds specifically to the element A in ARS1 in an ATP-dependant manner. ¾This complex also binds to other ARSs. The ORC remains bound to an ARS throughout the cell cycle and during replication becomes associated with other proteins –this triggers DNA synthesis. ¾Yeast mutants defective in any of the proteins of ORC are defective in DNA replication.

15

SV 40 origin of replication

A 65-bp region in the SV40 chromosome is sufficient to promote DNA replication both in animal cells and in vitro. Researchers have used mammalian proteins and plasmids carrying the SV40 origin to study the molecular mechanisms of DNA replication.

Common features of replication origins

Although the specific nucleotide sequences of replication origins from E.coli, yeast, and SV40 are very different, they share several properties: 9Replication origins are unique DNA segments that contain multiple short repeated sequences. 9These short repeat units are recognized by multimeric originbinding proteins. These proteins play a key role in assembling DNA polymerases and other replication enzymes on the sites where replication begins. 9Origin regions usually contain an AT-rich stretch. Origin-binding proteins control initiation of DNA replication by directing the assembly of replication machinery to specific sites on the chromosome.

16

General features of chromosomal replication - conclusions

1. The general features of chromosomal replication seem to apply with little modification to all types of cells. 2. DNA replication is semiconservative. 3. Once replication has started it continues until the entire genome has been duplicated. 4. It starts at origin. An origin “fires” ones and only ones during the cell cycle. 5. Replication is bidirectional. 6. At the place of the replication start (origin) helix unwinds and creates two replicational forks.

The DNA replication machinery

¾DNA polymerases are unable to melt duplex DNA (I.e. break certain hydrogen bonds) in order to separate strands that are to be copied ¾All DNA polymerases so far discovered can only elongate a preexisting DNA or RNA strand, the primer; they can not initiate chains. ¾The two strands in the DNA duplex are opposite (5’→3 and 3’ →5’) in chemical polarity, but all DNA polymerases catalyze nucleotide addition at the 3’hydroxyl end of a growing chain – only 5’→3 direction.

17

DnaA protein initiates replication in E.coli

Genetic studies suggested that initiation of replication at oriC in E.coli is dependent upon protein coded by dnaA gene. DnaA protein binds with oriC. Although DnaA can bind to duplex E.coli origin DNA in the relaxed-circle form, it can initiate replication only when the DNA is negatively supercoiled. The reason – negative supercoiles are tightly wound and are easier to melt locally (thus providing a single-stranded template region) than DNA molecules w/o supercoiles. Supercoiling is controlled by enzymes called topoisomerases. Binding of DnaA to oriC 9-mers facilitates melting of duplex DNA, which occurs at oriC 13-mers. This process requires ATP and yields so called open complex.

18

DnaA protein initiates replication in E.coli

DnaA binds oriC. Genetic studies of recombinant E. coli pointed that DnaA binds oriC, forming initial complex, and melts DNA at 9-mers and 13-mers.

19

Further melting of the two strands in E.coli chromosome to generate unpaired template strands is mediated by the protein product of the dnaB locus - a helicase that is essential for DNA replication.

One molecule of DnaB, DnaB, a hexamer of identical subunits, subunits clamps around each of the two single strands in the open complex formed between the DnaA and oriC. This binding requires ATP and the protein product of the dnaC locus.

20

The function of DnaC is to deliver DnaB to the template. One DnaB hexamer clamps around each single strand of DNA at oriC, forming the prepriming complex. DnaB is a helicase, helicase and the two molecules then proceed to unwind the DNA in opposite directions away from the origin.

DnaB is a helicase that melts duplex DNA Helicases constitute a class of enzymes that can move along a DNA duplex utilizing the energy of ATP hydrolysis to separate the strands. SSB protein - binds ssDNA Helicases exhibit directionality with respect to unwinding reaction. DnaB moves along the single strand of DNA to which it binds in the direction of it’s free 3’ end – it unwinds DNA 5’→3’ direction. DnaB, like many other proteins that act on DNA, is processive. Because it forms the clamp around ssDNA DnaB does not fall off until it reaches the end of the strand or is “unloaded” by other protein. Other kinds of helicases unwind in opposite direction, moving along the strand to which they are bound toward the free 5’ end.

21

E. coli primase catalyzes formation of RNA primers for for DNA synthesis

E. coli primase catalyzes formation of RNA primers for for DNA synthesis

Primase Catalyzes the formation of an RNA strand, complementary and antiparallel to a single DNA strand: oRNA strand grows 5'--> 3' ocomplementary to the DNA, read 3'-->5' Process: •Primer --> a short length of RNA-DNA duplex (about 10 nucleotides in length) 9DNA polymerase attaches to the duplex 9DNA polymerase forms a new DNA strand, starting at the 3'-end of the RNA strand.

22

E. coli primase catalyzes formation of RNA primers for for DNA synthesis

9The primers used during DNA replication in eukaryotes and prokaryotes are short RNA molecules whose synthesis is catalyzed by the RNA polymerase primase. 9Primase is usually recruited to a segment of single-stranded DNA by first binding to DnaB hexamer already attached at that site. The term primosome is now generally used to denote a complex between primase and helicase, sometimes with other proteins. 9In initiation of E. coli DNA replication, a primosome is formed by binding of primases to DnaB in prepriming complex. 9After bound primases synthesize short primer RNAs complementary to both strands of duplex DNA , they dissociate from the single stranded template.

E. coli primase catalyzes formation of RNA primers for for DNA synthesis

23

Replication, Okazaki fragments

DNA replication is continuous on the leading strand (1 primer); and discontinuous on the lagging strand – many primers. When newly formed fragment approaches the 5’ end of the other one DNA polymerase I takes over. It has exonuclease activity – removes RNA primer and fills the gap by adding deoxynucleotides.

Steps in the discontinuous synthesis of the lagging strand: strand this process requires multiple primers, two DNA polymerases, a ligase that joins the 3’ hydroxyl end of one Okazaki fragment with the 5’ phosphate of the adjacent fragment.

24

Ligation reaction:

During this reaction ligase transiently attaches covalently to the 5’ phosphate on one stand, thus activating the phosphate group. E. coli DNA ligase uses NAD+ as a cofactor, generating NMN and AMP. Bacteriophage T4 ligase, commonly used in DNA cloning, uses ATP, generating PPi and AMP.

Polymerases

9DNA polymerases are important enzymes involved in DNA replication. 9Three polymerases have been purified from E.coli. 9In addition to important role in filling the gaps between Okazaki fragments, DNA polymerase I is the most important enzyme for gap filling during DNA repair. 9DNA polymerase II functions in the inducible SOS response; this polymerase fills the gap and appears to facilitate DNA synthesis directed by damaged templates. 9DNA polymerase III catalyzes chain elongation at the growing fork of E. coli.

25

DNA polymerase I

1957 – Arthur Kornberg isolated an enzyme (DNA polymerase I) from E. coli that was able to direct DNA synthesis in vitro. Major requirements for in vitro DNA synthesis were: 1. All four deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP = dNTP). 2. Template DNA

DNA Polymerases II and III

1969 – Peter DeLucia and John Cairns discovered a mutant strain of E. coli that was deficient in polymerase I activity. Observation: the mutant strain duplicated its DNA and reproduced itself but cells are highly deficient in DNA repair (UVsensitive). Conclusions: 1. At least one more enzyme is able to replicate E. coli DNA. 2. DNA polymerase I may serve a secondary (at least for replication) function which is associated with DNA fidelity. Two other unique DNA polymerases have been isolated

26

Role of polymerases in vivo

Polymerase I : -removes the RNA primer; -fills the gaps that naturally occur as primers are removed; -has proofreading function. Polymerase II: -is involved in UV-damaged DNA repair; -has proofreading function. Polymerase III: -is the most replication relevant polymerase; -has proofreading function.

Properties of Three Bacterial DNA Polymerases

Initiation of chain synthesis 5’-3’ polymerization 3’-5’ exonuclease activity 5’-3’ exonuclease activity Molecules of polymerase/cell Synthesis from Intact DNA Primed single strands Primed single strands plus SSB Protein In vitro chain elongation rate Mutation lethal?

I + + + 400

II + + ?

III + + 15

+

-

-

+ 600 +

? -

+ 30000 +

27

DNA Polymerase III Holoenzyme

9 The DNA polymerase III holoenzyme is a very large (>600 kDa), highly complexed protein composed of 10 different polypeptides. The so called core polymerase is composed of 3 subunits. 9 The α subunit contains active site for nucleoride addition, and the ε subunit is a 3’-5’ exonuclease that removes incorrectly added (mispaired) nucleotides at the end of growing chain. The function of θ is still unknown. 9 The central role of the remaining subunits is to convert the Polymerase III from distributive enzyme which falls the template after forming short stretches of 10-50 nucleotides to processive enzyme which can form stretches of up to 5 x 105 nucleotides before being released from the template.

DNA Polymerase III Holoenzyme

The key to the processive activity of polymerase III is β subunit that forms a donut-shaped dimer around the DNA duplex and then associates with and holds the catalytic core polymerase near the 3’ terminus of growing strand. Once associated with DNA , the β subunit functions like a “clamp” which can slide freely along the DNA as the associated core polymerase moves. In this way active sites of core polymerase remain near the growing fork and the processivity of the enzyme is maximized.

28

DNA Polymerase III Holoenzyme

Out of the six remaining subunits 5 (γ,δ, δ1,χ and ψ) form socalled γ complex that mediates two essential tasks: 1) Loading of β subunit clamp onto the duplex DNA-primer substrate in a reaction that requires hydrolysis of ATP; 2) unloading of β subunit clamp after a strand of DNA has been completed. Loading and unloading of the β subunit clamp require opening of the clamp ring, but exactly how the γ complex does it is still unknown. The final τ subunit acts to dimerize two core polymerases and is essential to coordinate the synthesis of leading and lagging strands.

Subunits of DNA Polymerase III Holoenzyme

Subunit Function Groupings α 5’-3’ polymerization “Core” enzyme: Elongates polynucleotide ε 3’-5’ exonuclease chain and proofreads ?? θ γ Loads enzyme on δ template (Serves δ’ γ complex as clamp loader) χ ψ β τ

Sliding clamp structure (Processivity Factor) Holds together the two core polymerases at the replication fork

29

Subunits of DNA Polymerase III Holoenzyme

Space-filling model based on X-ray crystallographic studies of the dimeric β subunit binding to DNA duplex. Two β subunits (red and yellow) form a donat-shapes clamp. That remains tightly bound to a closed circular DNA molecule bur readily slides off.

Schematic diagram of proposed association of the core polymerase with the β subunit clamp at the primer-template terminus. This interaction keeps the core from falling off the template and positions is near the point of nucleotide addition.

Leading and lagging strands are synthesized concurrently Leading and lagging strands are linked together by a τ subunit dimer. Two molecules of core polymerase are bound at each growing fork: one at leading strand, the other one at lagging strand.

1) A single DnaB helicase moves along the lagging strand towards its 3’ end and melts the duplex DNA at fork. 2) One core polymerase (core1) quickly adds nucletides at 3’ end of the leading strand as its single-stranded template is uncovered by the helicase action of DnaB. This leading strand polymerase, together with its β subunit clamp remains bound to DNA, synthesizing leading strand continuously.

30

Leading and lagging strands are synthesized concurrently 3) Second core polymerase (core2) synthesise the lagging strand discontinuously as an Okazaki fragment. The two core polymerases are linked by a dimeric τ protein.

4) As each segment of the ss template for the lagging strand is uncovered, it becomes coated with the SSB protein and forms a loop. Once synthesis of an Okazaki fragment is completed, the lagging strand polymerase dissociates form DNA but core remains bound to the τ dimer. The released polymerase subsequently rebinds with the assistance of the another β clamp in the region of the other Okazaki fragment.

Leading and lagging strands are synthesized concurrently

9Two molecules of core polymerase are bound at each growing fork: one at leading strand, the other one at lagging strand. 9The core polymerase synthesizing the leading strand moves, together with its β subunit clamp, along its template in the direction of movement of the fork, elongating the leading strand. 9It follows closely the movement of DnaB protein that melts the duplex DNA of the fork. 9Since the core polymerase remains attached to the duplex DNA the leading strand is synthesized continuously.

31

Leading and lagging strands are synthesized concurrently 9The other core-polymerase molecule, which elongates the lagging strand, moves with its its β subunit clamp in the direction opposite to the fork movement. 9As elongation of the lagging strand proceeds, the size of the DNA loop between the fork and this core polymerase increases. Eventually core polymerase synthesizing the lagging strand will complete an Okazaki fragment , then it dissociates from the DNA template but the τ-subunit dimer remains to link it to the fork proteins.

Leading and lagging strands are synthesized concurrently 9Simultaneously, primase binds to the site adjacent to the DnaB helicase on the single-stranded segment of the lagging strand template and initiates synthesis of another RNA primer. 9The resulting DNA primer complex attracts another β subunit clamp to this segment of lagging strand template, followed by rebinding of the core polymerase, which is still attached to the complex. This polymerase then proceeds to elongate the RNA primer into another Okazaki fragment. As each Okazaki fragment nears completion, the RNA primer is remover by the 5’→3’ exonuclease activity of DNA polymerase I. 9This enzyme also fills the gaps between the lagging strand fragments, which are ligated together by DNA ligase.

32

Leading and lagging strands are synthesized concurrently 9Although the two core polymerase molecules are linked by τsubunit dimer, they are oriented in opposite directions. 9Thus, the 3’ growing ends of both leading and lagging strands are close together but offset from each other. For this reason the point of the template from which the lagging strand is being copied is displaced from the point in the template at which leading strand copying is occurring. 9 Nonetheless, the two core polymerases can add deoxyribonucleotides to the growing strands at the same time and rate, so that leading and lagging strand synthesis occur 9s concurrently.

Leading and lagging strands are synthesized concurrently

9One τ-subunit also contacts the DnaB helicase at the fork. This interaction strongly increases normally slow unwinding activity of the helicase. 9 Thus, there is a physical and functional link between the two major replication machines at the fork – the two core polymerases and the primosome complex of DnaB and primase.

33

Synthesis of leading and lagging strands

Cycling of polIII complex

34

Replication fork in E. coli

Replication in eukaryotics is very similar to that in prokaryotic cells.

Because eukaryotic cells have more DNA they also have more origins of replication. A mammalian cell for example has about 1 x 109 basepairs of DNA. There is an origin of replication about every 30,000 basepairs of DNA, though the structure of these sites is not clearly understood. The DNA synthesis is also much slower than in prokaryotic cells because of the chromatin proteins, synthesis is about 100 nucleotides per second.

35

Mammalian DNA polymerases Much less is known about mammalian proteins involved in DNA replication. It had been thought that polymerase α synthesizes the lagging strand because of its low processivity. Polymerase δ is much more processive than α, as it is assoxiated with the PCNA clamp. PCNA is enriched in proliferating cells and enhances the processivity of pol δ about 40 times. Pol β is not processive at all – it can do just 1 nucleotude – fits to its repair enzyme role. Pol γ if found only in mitochondria.

Probable roles of eukaryotic polymerases Polymerase α - priming replication on both strands Polymerase δ - elongation of both strands Polymerase β - DNA repair Polymerase ε - DNA repair Polymerase γ - replication of mitochodrial DNA

36

Properties of mammalian DNA polymerases Mammalian polymerases 5’-3’ polymerization 3’-5’ exonuclease proofreading activity Synthesis from RNA primer DNA primer Associated DNA primase Sensitive to aphidicolin (inhibitor of cell DNA synthesis) Cell location Nuclei Mitochondria

α + -

β + -

γ + +

δ + +

ε + +

+ + +

+ -

+ -

+ + +

+ +

+ -

+ -

+

+ -

+ -

Eukaryotic replication machinery is generally similar to that of E. coli

9Like DNA replication in E. coli, eukaryotic DNA replication occurs bidirectionally from RNA primers made by a primase, synthesis of the leading strand is continuous, while synthesis of the lagging strand is discontinuous. 9In contrast to E. coli two distinct polymerases, α and either δ or ε, function on the eukaryotic growing fork.

37

9SV40 DNA can replicate in mammalian cells. 9Replication is initiated by binding of a virus-encoded protein called T-antigen to the SV 40 origin of replication. This multifunctional complex binding melts DNA through its helicase activity. Opening of the duplex at the SV40 origin also requires ATP and replication protein A (RPA), a host cell single stranded binding protein, with a function similar to that of SSB of E.coli. 9One molecule of polimerase α (Pol α) tightly associates with primase, then binds to each unwound template strand.

Eukaryotic replication machinery is generally similar to that of E. coli

9The primases form RNA primers, which are elongated for a short stretch by Pol α, forming first leading strands, which grow from the origins in to different directions. 9The activity of Pol α is stimulated by replication factor C (RFC).

38

9PCNA (proliferating cells nuclear antigen) then binds to the primer-template 3’ termini, displacing Pol α from both leading strand templates and thus interrupting leading strand synthesis. 9Next Pol δ binds to PCNA at the 3’ ends of the growing strands. The association of Pol δ with PCNA increases the processivity of the polymerase so that it can continue the synthesis of the leading strand without interruption.

PCNA and DNA

PCNA is a trimer that forms a “ clamp” around duplex DNA.

39

PCNA and DNA

Eukaryotic replication machinery is generally similar to that of E. coli

9Thus the function of PCNA is highly analogous to that of the β subunit clamp of the E.coli polymerase III, as both proteins form rings around DNA. But, the amino acid sequences of them are different and β subunit clamp is a dimer and PCNA is a trimer. 9As melting of the duplex DNA, catalyzed by a hexameric form of Tag progresses further away from the origin, the primase-Pol α complex associates with melted template downstream from leading-strand primers.

40

9Synthesis of the lagging strand is then carried out by combined action primase and Pol α, along with RFC, Pol δ and PCNA while leading strand synthesis on the other side of the origin also proceeds. 9Finally, in eukaryotes as in E. coli topoisomerases play an important role in relieving torsional stress induced by growing fork movement and separating the strands.

A model for eukaryotic chromosome replication •Unwinding at origin of replication •DNA pol α-prim initiates DNA synthesis •PCNA, RF-C, pol δ, ε bind •Polymerase switch on lagging strand

41

Termination of DNA Replication

Several steps are involved in the termination of DNA replication: 1) Removal of RNA primers by DNA polymerase •When DNA polymerase encounters an RNA primer in its path, its proofreading mechanism recognizes that it is not DNA-DNA duplex and: oremoves the RNA primer, one ribonucleotide at a time (exonuclease activity) oinserts a deoxyribonucleotide, complementary to the base of the template strand. orepeats the process until all of the RNA is removed and replaced with DNA double strand. •This process occurs: oon the lagging strand, at the beginning of Okazaki fragments. oon the leading strand at the replication origin.

2) Closing the DNA-DNA gaps Problem: Removal of RNA primers by DNA polymerase leaves gaps between the 5'-P end of one nucleotide and the 3'-OH end of another. Therefore: There is no high energy phosphate bond to supply energy to close the gap with a phosphodiester bond. Solution: DNA ligase 9Unlike bacterial chromosomes, that are circular, eukaryotic chromosomes are linear and carry specialized ends called telomeres.

42

Termination in prokaryotes

Replication has a beginning and an end. In bacterial replication two forks approach each other in the terminus region – which contains 22-bp terminator sites that bind specific proteins. In E. coli the terminator sites are called TerA-TerF (E,D,A,C,B,F). They are the binding sites for the Tus proteins (terminus utilization substance). Sequences must be disentangled.

Termination in eukaryotes Telomeres – ends of eukaryotic chromosomes are composed of GC-rich sequences. The GC-rich strand of a telomere is added at the very 3’ end of DNA strands, in a semiconservative replication, by an enzyme telomerase. The exact repeat of telomere is species specific. In vertebrates, including humans, it is TTAGGG/AATCCC Telomerase add many repeated sequences at the ends of chromosome. Telomerase contains short RNA that serves as a template for telomere synthesis. Priming can then occur within these telomeres to make a C rich strand

43

NOTE: The C-rich telomere strands is synthesized by ordinary RNAprimed DNA synthesis, like the lagging strand in conventional DNA replication. This mechanism ensures that chromosome ends can be rebuilt and not shorten with each round of replication.

Telomeres and telomerase.

44

Forming of telomeres

Telomerase prevents progressive shortening of lagging strands during eukaryotic DNA replication

9Telomeres consist of repetitive oligomeric sequences. The need for a specialized region at the ends of chromosomes is apparent – all known DNA polymerases elongate DNA chains from the 3’ end, and all require DNA or RNA primer. 9As growing fork approaches the end of the linear chromosome, synthesis of leading strand continues to the end of DNA template strand; the resulting completely replicated daughter DNA double helix then is released.

45

Telomerase prevents progressive shortening of lagging strands during eukaryotic DNA replication

9However, because the lagging strand is copied discontinuously, it can not be replicated in its entirety. 9When the final primer is removed there is no upstream strand onto which DNA polymerase can build to fill the resulting gap. 9Without some special mechanism the daughter DNA strand resulting from lagging strand synthesis would be shortened at each cell division.

Telomerase prevents progressive shortening of lagging strands during eukaryotic DNA replication

9The enzyme that prevents this progressive shortening of lagging strand is a modified reverse transcriptase, called telomerase. 9It can elongate the lagging strand template from its 3' hydroxyl end. This unusual enzyme contains a catalytic site that polymerizes deoxyribonucleotides directed by an RNA template, and the RNA template itself is brought to the site of calalysis as part of the enzyme.

46

9The repetitive sequence added by telomerase is determined by the RNA associated with the enzyme, which varies between the telomerases from different sources. 9Once the 3’ end of the lagging strand is sufficiently elongated, synthesis of the lagging strand can take place, presumable from additional primers. 9For cells in many organisms, telomere length is increased many times over early in development.

Interestingly

‰Most human somatic cells replicate in the absence of the telomerase activity and thus gradually consume the telomeric repeats added earlier in development. ‰The progressive shortening of the chromosome ends and eventual loss of genetic information that results has been linked to cell death. ‰It has been suggested that life span is determined by the number of telomeres with which the individual starts. ‰Indeed, the reverse relationship between the age and telomere length has been observed.

47

Mechanism of action of telomerase

To summerize –eukaryotic and prokaryoric DNA replication scenarios are very similar When DNA replicates, many different proteins work together to accomplish the following steps: 1.The two parent strands are unwound with the help of DNA helicases. 2 . Single stranded DNA binding proteins attach to the unwound strands, preventing them from winding back together.

The major types of proteins, which must work together during the replication of DNA

3 The strands are held in position, binding easily to DNA polymerase, which catalyzes the elongation of the leading and lagging strands. (DNA polymerase also checks the accuracy of its own work!).

48

4. While the DNA polymerase on the leading strand can operate in a continuous fashion, RNA primer is needed repeatedly on the lagging strand to facilitate synthesis of Okazaki fragments. DNA primase, which is one of several polypeptides bound together in a group called primosomes, helps to build the primer. 5. Finally, each new Okazaki fragment is attached to the completed portion of the lagging strand in a reaction catalyzed by DNA ligase.

Summary – DNA replication machinery

¾The enzymes and other protein factors that carry out DNA replication in E. coli and in eukaryotic cells are analogous, suggesting that the biochemical mechanism of DNA replication is similar in all cells. ¾The enzymatic events at the growing fork are a consequence of two properties of the DNA double helix and two of polymerases: the double helix contains two antiparallel strands and the two strands are interwound, so they can not simply be melted along the entire length at once. ¾Dna polymerases require a nucleic acid primer –either a DNA or an RNA molecule to begin synthesis and all DNA chain growth occurs by nucleotide addition at the 3’ end.

49

Summary – DNA replication machinery

¾In all cells, one new DNA strand, the leading strand is synthesized continuously in the direction of movement of the growing fork by elongation from the 3’ end of the RNA primer base-paired to a template strand.Synthesis of the other strand, the lagging strand, occurs in the direction opposite to the overall direction of the replication fork movement from a series of short RNA primers formed on the second template strand. The resulting segments of RNA plus DNA are called Okazaki fragments. After the primers are removed and the gaps are filled, they are joined. ¾Initiation of DNA replication in E. coli occurs by binding of DnaA to oriC, followed by attachment of DnaB, a helicase that melts DNA at the fork. Association of primase with this complex forms a primosome. After primer synthesis primase dissociates.

Summary – DNA replication machinery

¾E. coli polymerase III catalyzes nucleotide addition to both leading and lagging strands. DNA polymerase I removes the RNA primers from Okazaki fragments and fills the gaps on the lagging strand. Finally, DNA ligase joins the Okazaki fragments. ¾Eukaryotic proteins that replicate SV40 DNA in vitro exhibit similarities with E. coli replication proteins. A viral protein called T antigen functions similarly to the DnaB helicase, and host-cell PCNA is similar to the β-subunit clamp associated with E. coli DNA polymerase III. However, two distinct mammalian polymerases, α and δ or ε, function on eukaryotic growing fork.

50

Summary – DNA replication machinery

The processivity of DNA polymerase is essential for efficient polymerization and is facilitated by their association with the βsubunit clamp in E. coli and PCNA in eukaryotes. Telomerase, a reverse transcriptase that contains an RNA template, adds nucleotides to the 3’end of the lagging strand template and thus prevents shortening of lagging strands during replication of linear DNA molecules such as those of eukaryotic chromosomes.

Reading – chapters 20,21, excluding DNA repair

51