Fig. 10-CO, p.240

Prokaryotic cells divide by pinching in two

Learning Objectives 1. What Is the Flow of Genetic Information in the

Cell?

2. What Are the General Considerations in the

Replication of DNA?

3. How Does the DNA Polymerase Reaction Take

Place?

4. Which Proteins Are Required for DNA

Replication?

5. How Is DNA Replicated in Eukaryotes?

Fig. 10-1, p.241

The Central Dogma The flow of genetic information in biological

systems.

It states that in all living organisms, the

genetic information is stored in chromosomal

DNA. The flow of genetic information occurs

in one direction from DNA RNA Protein

Fig. 10-18, p.256

The eukaryotic cell cycle

DNA Replication

In this process the two polynucleotide chains

of DNA are separated and each is copied

in a complementary manner to produce

daughter polynucleotide chains .

Each daughter DNA molecule will contain

one polynucleotide comes from the

parental DNA and the other chain is newly

formed ( semiconservative replication)

Fig. 10-3, p.243

Experimental evidence for semiconservative replication. Meselon-Stahl Experiment

1958 ( In Prokaryotes )

Fig. 10-2, p.242

DNA replication must occur in order to faithfully

transmit genetic material to the progeny of any cell or

organism.

DNA replication takes place during S phase of the cell

cycle.

Transcription is the process by which the information

contained in a section of DNA is transferred to a newly

assembled piece of messenger RNA (mRNA).

Translation a process in which proteins can be

synthesized using the information in mRNA as a

template .

• In eukaryotic genome there is no similar linear relationship between genetic information carried in DNA and proteins expression, which was observed in prokaryotic system

• Occasionally, genetic information flows from RNA to DNA (in reverse of normal transcription). This is known to occur in the case of retroviruses, such as HIV.

DNA replication

• In prokaryotes like E. coli , the replication

starts at fixed point called the origin and

proceeds bidirectional along the

chromosomal DNA (both directions) until

the whole circular DNA is completely

replicated. By then the replication process

is terminated at certain point on DNA.

Fig. 10-4a, p.244

Fig. 10-5a, p.245

Fig. 10-5b, p.245

Mechanism of DNA replication in Prokaryotes

• Relaxation of complex super structure of

chromosomal DNA

• Inside bacteria the circular double helix DNA is present in super helical form in which the DNA is further twisted in more circles. The relaxed double helix is called a secondary structure and the complex super helix called tertiary structure. The super helix is needed to pack the DNA in small space inside the cell but it is not suitable form for starting DNA replication. So, the first step in starting DNA replication is the relaxation of super coiled DNA by making small cut (nicking) with the enzyme Topoisomerase (Gyrase).

DNA replication involves 3 stages

Initiation, elongation and termination.

1.Initiation:

• Initiation of DNA replication starts by the binding

of the initiator factor protein DnaA at the initiation

point in DNA, which recognizes a repeat 9 A-T

base pair rich region. The binding of this protein

helps in local unwinding double helix DNA at the

initiation point by the enzyme helicase. After

local unwinding the two single strands DNA are

kept separated and not folded back again by

single strand binding proteins.

Formation of bubble origin point

• The separation of the two DNA strands

expand the DNA region at the origin point

to form a bubble shape area that help in

the incorporation of deoxynucleotides to

synthesis new DNA strands.

II. Elongation of new DNA

• At the bubble area, local unwinding and replication will grow in two opposite directions so that both polynucleotides are copied simultaneously .This polynucleotides growth form a Y-shaped replication fork at each direction site of DNA replication (2 replication forks).

• Since, the DNA synthesis occurs in 5′ to 3′ direction, one strand called the leading strand, can be synthesized continuously while the other called the lagging strand, must be synthesized backward discontinuously in short fragments (Okazaki fragments) which are later joined to make one long piece.

Properties of prokaryotic DNA polymerase

enzymes

• There are 3 different types of prokaryotic DNA

polymerases ,called pol I,pol II, pol III.Only pol

I and III are involved in DNA replication while

pol II function is limited to DNA repair of

damaged DNA.The pol I enzyme has slower

activity than pol III enzyme.

• Each DNA polymerase enzyme has two types

of activities, polymerization and nuclease

activities.

Table 10-1, p.246

t

Fig. 10-7, p.246

The dimer of β-subunits of DNA polymerase III bound to DNA

1) Polymerization activity

• Add deoxynucleotids ( as monophosphate)

from triphosphates deoxyncleotides , using

energy liberated from the hydrolysis of

pyrophosphates (the process is called

polymerization).The monophosphate

deoxynuleotides are connected by

phosphodiester linkage in 53 direction.

The building blocks for this process

are 5'-ribonucleoside triphosphates,

and pyrophosphate released as each

phosphodiester bonds made.

RNA primer

DNA polymerases cannot initiate synthesis of a

complementary strand of DNA on a totally single-

stranded template. Rather, they require an RNA

primer that is, a short, double-stranded region

consisting of RNA base-paired to the DNA

template, with a free OH-group on the 3'-end of

the RNA strand .This OH group serves as the first

acceptor of a nucleotide by action of DNA

polymerase. In de novo DNA synthesis, that free

3'-hydroxys provided by the short stretch of RNA,

rather than DNA.

Primase

A specific RNA polymerase, called primase

synthesizes the short stretches of RNA

(approximately ten nucleotides long) that are

complementary and antiparallel to the DNA

template. In the resulting hybrid duplex, the U in

RNA pairs with A in DNA. These short RNA

sequences are constantly being synthesized at the

replication fork on the lagging strand, but only one

RNA sequence at the origin of replication required

on the leading strand.

Primosome

Prior to the beginning of RNA primer synthesis on

the lagging strand, a prepriming complex of

several proteins assembled and binds to the

single strand of DNA, displacing some of the

single-stranded DNA-binding proteins. This

protein complex, plus primase is called the

primosome. It initiates Okazaki fragment

formation by moving along the template for the

lagging strand in the 5’ 3’ direction, periodically

recognizing specific sequences of nucleotides

that direct it to create an RNA primer thats

synthesized in the 5’ 3’ direction (antiparallel to

the DNA template chain).

• 2) Exonuclease activity:

Repeated cutting of single nucleotide from the

terminal end of a polynucleotide

• 3-Exonuclease activity: Removal of

polynucleotide sequence in 35 direction

from the terminal end associated with pol III

activity.

• 5- Exonuclease activity: Removal of

polynucleotide sequence in 53 direction

from the terminal end, associated with pol I

activity.

Fig. 10-11, p.250

DNA polymerase I proofreading removes nucleotides from the

3’ end of the growing DNA chain.

Fig. 10-12a, p.252

The 5’3’ exonuclease activity of DNA polymerase I can remove

up to 10 nucleotides in the 5’ direction downstream from a 3’-OH

single –strand nick

Mechanism of DNA synthesis at each

Replication Fork

1)-Leading strand

RNA primase enzyme creates a short primer

RNA with free 3' end ( 10 RNA nucleotide

sequence) .

DNA polymerase III enzyme - uses a single

parent strand of DNA as a template to add new

nucleotides to the 3' OH end of initially

incorporated RNA primer.

• The addition is continuous according to the base pairing rule.

• If a mismatch is accidentally incorporated, the polymerase is inhibited from further extension. Proofreading removes the mismatched nucleotide and extension continues. The mismatched nucleotides are remove by the exonuclease activities of DNA polymerase III.

• Later , DNA polymerase I removes the RNA primers and replaces them by DNA pieces leaving a small gap of free 3' OH and 5’ OH ends to be sealed by DNA ligase .

2)-Lagging strand

DNA polymerase is unable to work directly on the lagging strand because it lacks a free 3- OH end on the existing DNA strand.

The new strand is synthesized in short discontinuous segments, each segment consists of RNA primer formed by primase and replicated DNA piece (Okazaki fragments) about 100-200 DNA nucleotides in length formed by DNA polymerase III similarly to the leading strand except that the addition takes place in backward direction

Later the polymerase I removes the RNA primers and replace them by DNA fragments leaving gaps .

Ligase enzyme seals the gaps as described before..

Reiji Okazaki

(1930 –1975) • was a pioneer Japanese molecular biologist, known for his

research on DNA replication and especially for describing the

role of Okazaki fragments which he discovered working with

his wife Tsuneko in 1966.

• Okazaki was born in Hiroshima, Japan. He graduated in 1953

from Nagoya University, and worked as a professor there

after 1963. He died of leukemia (due to Atomic bombings of

Hiroshima) in 1975 at the age of 44; he had been

heavily irradiated in Hiroshima when the first atomic

bomb was dropped. His wife, Tsuneko, won the L'Oréal-

UNESCO Awards for Women in Science in 2000 for her work.

Final results of replication at the

fork level

III.Termination:

Termination requires that the progress of the DNA replication fork must stop at a specific locus on DNA. This process involves the interaction between two components:

(1) A termination site sequence in the DNA

(2) A protein which binds to this sequence to physically stop DNA replication.

In bacteria this protein is named the DNA replication terminus site-binding protein.

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks meet each other on the opposite end of the parental chromosome.

In E. coli the chromosomal DNA replication takes about 40 minutes to replicate the 4000 kb size of DNA. Therefore each fork replicates 2000 kb in 40 min. or ~ 50 kb/min or ~1000 bases/sec.

p.251a

Why Does DNA Contain Thymine & Not Uracil ?

The incorporation of thymine instead of uracil helps ensure that the DNA is replicated

faithfully.

p.251b

Fig. 10-10, p.249

General features of a replication fork

Table 10-3, p.250

END Part I

Eukaryotic DNA replication

• Eukaryotic genome is more complex than

prokaryotic genome. For example human

genome is composed of 30,000-40,000

genes, and each gene is segmented into two

types of DNA pieces, exons and introns ,Each

gene has on average between 5 and 8 exons,

8000 base-pairs.

• Exon: DNA segment which after transcription to RNA codes directly to peptide units of a polypeptide, i.e which `is expressed in protein' (200 base-pairs on average, in human genome)

• Intron: DNA segment which is not directly expressed for protein, involved in regulation, splicing and other unknown functions each 2000 bp's on average, in human genome.

Cell cycle in eukaryotes

• Actively dividing eukaryote cells pass through a series of stages known collectively as the cell cycle: involving interphase stage between each two mitosis. The interphase stage itself includes 3 phases represented by two gap phases (G1 and G2); and S (for synthesis) phase, in which the genetic material is duplicated. The two gaps are preparative periods for cell division and an opportunity for the cell to make decision whether to go in to division or not. In the M phase a nuclear division takes place before it is followed by cell division that occurs in cytokinesis

• In the S phase. DNA synthesis replicates the genetic material and each chromosome becomes having two sister chromatids. Therefore, only in this period of the interphase that DNA replication occurs which is a necessary step before the cell decides to go in to division(without DNA replication there is no cell division).In human the S period is carried out for about 8 hours in an average total cell cycle period of around 20 hours.

• Eukaryotic DNA synthesis is similar to

synthesis in prokaryotes, except for some

complexity. In eukaryotic cells:

• there is more DNA than prokaryotic cells

• the chromosomes are linear

• the DNA complexes with proteins

Eukaryotic replication initiated at many

points

• Because the eukaryotic genome is so large (about 100 times the size of bacterial DNA), it would take days to replicate the whole length of eukaryotic chromosome using the same single initiation point as in prokaryotes.

• Therefore, many initiation points ( about 10,000 in human) are found in each eukaryotic chromosome instead of one, with replication forks moving in opposite directions away from each initiation point until they meet in the middle between each two initiation points. The initiation point is called a replicons which do not need specific termination sequences.

Fig. 10-4b, p.244

• Not all replicons are activated

simultaneously. Rather, clusters of 20-80

adjacent replicons are activated

throughout S phase until the whole

chromosome is completely replicated.

• The rate of eukaryotic DNA replication is

much slower than E. coli, with only 100-

200 nucleotides bases/sec are replicated

in eukaryotic Okazaki fragments.

However, the majority of replication forks

results in the whole genome being

replicated in only about 8 hours. Histones

for packaging the DNA are synthesized

simultaneously with DNA replication to

bind the new DNA.

Enzymes involved in eukaryotic DNA

replication

1.DNA Polymerase α

• Initiation the synthesis of RNA primer (about 20-30 ribonucleotides) then adds DNA to the RNA primers

• It has low processivity (efficiency) of DNA synthesis and has no 35 exonuclease activity .

2.DNA Polymerase δ

• The principal DNA polymerase in eukaryotic DNA replication which has 35 exonuclease activity.

• When it complexes with PCNA (Proliferating Cell Nuclear Antigen) it becomes highly processive.

Additional Proteins Involved in

Eukaryotic DNA Synthesis

• DNA helicase: the enzyme which carries out partial unwinding of double helix DNA at the initiation point before the starting of DNA replication

• PCNA (Proliferating Cell Nuclear Antigen)

Provides high processivity to DNA Polymerase δ

• RPA (Replication Protein A)

• ssDNA-binding protein that facilitates the unwinding of the helix to create two replication forks.

• RFC (Replication Factor C) binds PCNA at the end of the primer

• FEN1/RTH1 (flap endonuclease 1/RAD two homologue 1) exonuclease complex

Leading strand synthesis

1) Starts with the primase activity of DNA Pol α to put down RNA primer in 5’ 3’-direction

2) The same enzyme adds a piece of DNA to the primer

3) RFC binds PCNA at the end of the primer

4) PCNA displaces DNA Pol α.

5) DNA polymerase δ binds to PCNA at the 3’ ends of the growing strand to carry out polymerase switching to highly processive DNA synthesis activity.

The RFC mediates the polymerase switching by helping in the

a) Assembly of PCNA

b) Removal of DNA Pol α

c) Addition of DNA Pol δ

Lagging strand synthesis

1) Starts off the same way as leading strand synthesis

2) RNA primers synthesized by DNA polymerase α every 50

nucleotides and consist of 20-30 nucleotides RNA

3) DNA polymerase δ switching as before to extend the RNA

primers and generating Okazaki fragments

4) When the DNA Pol δ polymerizes the RNA primer of the

downstream Okazaki fragment, RNase H1 removes all but the

last RNA nucleotide of the RNA primer

5) The FEN1/RTH1 exonuclease complex removes the last RNA

nucleotide

6) DNA Pol δ fills in the gap as the RNA primer is being

removed

7) DNA ligase joins the Okazaki fragment to the growing strand

Telomeres problem during human DNA

replication

• Telomeres present at the ends of linear

chromosomal DNA and consist of long

area of short repeating sequences

TTGGGG ->->->->- to protect the integrity

and stability of human chromosomes.

During DNA synthesis these chromosome

ends cannot be replicated with DNA

polymerase.

This sequence of TTAGGG is repeated

approximately 2,500 times in humans. In

humans, average telomere length declines

from about 11 kilobases at birth to less

than 4 kilobases in old age, with average

rate of decline being greater in men than

in women.

Telomeres are found at the termini of

chromosomes. The end of a telomere inserts back

into the main body of the telomere to form a T-loop

• Since DNA can only be synthesized at the 3'-

end of a pre-exiting DNA or RNA chain,

there is no available mechanism for achieving

DNA synthesis all of the ways to the end of the

lagging strand. Once the primer in the last

Okasaki fragment is removed by a 5' to 3'

exonuclease it is not possible to replace it with

DNA. This is because the 5 - ends of the

lagging strands does not have enough space to

put a new primer with free 3'-hydroxyl group

and therefore it is not copied completely.

• After maturation of Okazaki fragments,

there is a primer gap

This problem does not occur with the

leading strand which can undergo complete

replication round. If this phenomenon is

repeated over many rounds of replication,

some of the chromosomes will gradually

develop major shortening in their ends.

Correction of the chromosome ends by

the enzyme Telomerase Telomerase enzyme is a ribonucleo protein complex containing

RNA-dependent DNA polymerase activity and 450-nucleotide

RNA. It can act as a reverse transcriptase enzyme by using its

own repetitive RNA sequence (AAAACCCC ) as a template to

add a repeat complementary sequence of TTAGGG to the 3-

OH end of leading strand in telomeres of human DNA. This

addition step by telomerase is repeated several times until an

extend 3- end of the DNA is formed. In this case the role of

telomerase enzyme is ended leaving gap in the 5- phosphate

end of the opposite lagging DNA strand .This gap will be filled

later by primase (adding short RNA primer ) and combined

actions of DNA polymerase and ligase activities..

• Some somatic cells lack telomerase

activity and therefore, their telomeres get

shorter with each cell division (About 50

bases are lost from each telomere every

time a cell divides) which may end up with

cell death. To the contrary cancer cells

may have high activity of telomerase

enzyme that increases their survivals,

• Telomerase: Terminates the process of

DNA replication only at the telomere ends

of chromosomal DNA by adding many

repeat units that can not be recognized by

the replication complex.

p.259a

p.259b

p.258a

p.258b

Telomere replication (asterisks indicate sequences at the 3’ end that cannot

be copied by conventional DNA replication

Model for initiation of the DNA replication cycle in

eukaryotes

ORC is present at the replicators throughout the cell

cycle. The pre-replication complex (pre-RC) is

assembled through sequential addition of the RAP

(replication activator protein) & RLFs (replication

licensing factors ) during a window of opportunity

defined by the state of cyclin-CDKs.

Phosphorylation of the RAP,ORC,and RLFs triggers

replication.

After initiation, a post-RC state is established, and the

RAP & RLFs are degraded.

Fig. 10-19, p.257

Model for initiation of the DNA replication cycle in eukaryotes

Fig. 10-21, p.261

The basics of the eukaryotic replication fork

Table 10-4, p.257

Table 10-5, p.260

END

Part II