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DNA replication Flashcards

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1
Q

DNA replication

A

DNA replication is the synthesis of DNA
Process by which DNA molecules are copied

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2
Q

Why DNA replication is important

A
  • Central role in ensuring the faithful transmission of genetic information from one generation to the next,
  • Mutations are generated during DNA replication.
  • DNA replication and cell division allow an organism to grow
  • DNA replication allows tissue to regenerate
  • Replication is important in the development of diseases such as cancer
  • The DNA replication process represents a potential target for inactivation of
    viruses that are not susceptible to metabolic inhibitors such as antibiotics
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3
Q

DNA replication in tissue regeneration

A

In some tissues regeneration does not occur because the DNA does not replicate.
Nervous tissue is an example of this. By understanding and manipulating the process we may eventually be able to control the process

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4
Q

DNA replication in development of diseases

A

Tumor formation is essentially uncontrolled DNA replication and cell division

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5
Q

DNA replication and viruses example

A

The use of AZT in the treatment of HIV infection is a prime example of this.
Inhibiting DNA replication has also been used to control plant virus diseases

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6
Q

DNA replication errors per bases

A

A single celled human zygote contains 6.4 billion base pairs.
The DNA from the single zygote cell divides to generate
human body made of ~37 trillion cells.
Imagine a consequence if there is an error as low as 1 per
million bases
~6400 errors in one cell?

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7
Q

Proposed DNA replication models

A

Conservative replication model
Dispersive replication model
Semiconservative replication mode

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8
Q

Semiconservative mode

A

After replication both daughter duplexes contain one parental and one new strand

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9
Q

Conservative mode

A

Photocopy
After replication one daughter duplex is the same as parental DNA but the another daughter duplex has both strands new

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10
Q

Dispersive mode

A

After replication both daughter duplexes contain double stranded segments of parental and new strands

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11
Q

Experiment that determined the correct mode of DNA replication

A

Meselson-Stahl experiment (1958)
Their strategy involved a technique that could differentiate between parent (old) and
daughter (new) DNA

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12
Q

Technique to distinguish between heavy and lighter DNA

A

Nitrogen atoms in DNA come from the source of nitrogen in the growth medium.
DNA can be labelled with 14N or 15N by providing appropriately marked nitrogen source of the growth medium.
In equilibrium density gradient
centrifugation, DNA with different
densities migrates at different rates thus showing different bands
Heavy DNA (15N) moves towards the bottom, light DNA (14N) remains closer to the top

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13
Q

DNA polymerase

A
  • Makes DNA
  • DNA polymerase can’t synthesize DNA without an existing free 3`OH end which
    is supplied by a primer; DNA synthesis is initiated with a primer
  • DNA polymerase uses dNTPs as substrates; NOT NTPs
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14
Q

Primer

A

a short oligonucleotide, is made of ribonucleotides not
deoxyribonucleotides as RNA is distinguishable from DNA- ensures primer is removed from strand

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15
Q

Requirements of replication

A
  • A template strand
  • Raw material: primer, nucleotides (dATP, dCTP, dGTP, dTTP)
  • Enzymes and other proteins
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16
Q

Process of DNA replication

A
  • The two strands of DNA must first separate so that a single strand can be used as a template.
  • An RNA primer provides the
    3`end for DNA polymerase to add bases complementary to the
    template strand.
  • DNA synthesis takes place only in 5-3 direction
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17
Q

3’-5’ exonuclease activity

A

Removes wrong base

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18
Q

5’-3’ exonuclease activity

A

Removes primer

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19
Q

Type I DNA polymerase

A

5’-3’ polymerase activity- yes
3’-5’ exonuclease activity- yes
5’-3’ exonuclease activity- yes
Function- removes and replaces primers

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20
Q

Type II DNA polymerase

A

5’-3’ polymerase activity- yes
3’-5’ exonuclease activity- yes
5’-3’ exonuclease activity- no
Function- DNA repair; restarts replication DNA halts synthesis

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21
Q

Type III DNA polymerase

A

Main
5’-3’ polymerase activity- yes
3’-5’ exonuclease activity- yes
5’-3’ exonuclease activity- no
Function- elongates DNA

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22
Q

Type IV DNA polymerase

A

5’-3’ polymerase activity- yes
3’-5’ exonuclease activity- no
5’-3’ exonuclease activity- no
Function- DNA repair

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23
Q

Type V DNA polymerase

A

5’-3’ polymerase activity- yes
3’-5’ exonuclease activity- no
5’-3’ exonuclease activity- no
Function- DNA repair; translesion DNA synthesis

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24
Q

Process of DNA replication (synthesis)

A
  • A template strand is required to synthesize a desired sequence of DNA.
  • DNA bases, complementary to the template strand, are added one by one at the 3` end
    of DNA; dNTPs act as a source of new nucleotides.
  • The 5phosphate group of the incoming dNTP attacks the 3-OH of the last nucleotide
    on the strand.
  • A phosphodiester bond links the two nucleotides; two phosphates are released.
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25
At the origin of replication
a part of DNA separates in two strands to expose the templates
26
RNA primers
synthesized by the enzyme primase
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Replication fork
* moves further on both ends * It allows synthesis of new RNA primer on the unreplicated part of the template. DNA polymerase now can add bases at the 3` end of the RNA chain * As the replication fork opens further in both directions, two strands get continuously synthesized while the other two remain unreplicated and wait for a new primer * new primers are synthesized in two strands (marked region ‘d’) and DNA gets synthesized
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Leading strand
* template is continuous * it does not require new primer at further opening of replication fork. * This template strand is called the leading strand
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Lagging strand
the enzyme cannot add bases at the 5` end so DNA synthesis re-starts at the fork; this happens each time DNA unwinds
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Discontinuous synthesis
involving the production of small fragments (called Okazaki fragments), is a slower process than the synthesis of the other strand
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DNA synthesis in bacteria
* Initiation * Unwinding - DNA helicase - Single strand binding proteins - DNA gyrase * Elongation - Synthesis of primers - DNA synthesis by DNA polymerase III - DNA synthesis by DNA polymerase I - DNA ligase * Termination
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Initiation
* DNA replication starts at a specific point called origin of replication. * Bacterial chromosomes are replicated through a single origin of replication. * In E. coli the oriC region is made of 245 bp where the initiator protein DnaA binds. * By binding of DnaA at oriC, a short region of DNA unwinds, that allows helicase and ssDNA binding proteins to bind
33
Unwinding
* The DNA synthesizing enzymes require single stranded DNA template. It is achieved with the help of a number of proteins that unwind and stabilize DNA in single stranded form. * These proteins include: - DNA helicase (DnaB) - Single-strand-binding proteins - DNA gyrase
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DNA helicase (DnaB)
it breaks the hydrogen bonds between two strands
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Single-strand-binding proteins
they keep single stranded DNA stable
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DNA gyrase
a topoisomerase II that controls the supercoiling, it reduces the torque that builds up ahead of the replication fork
37
Nalidixic acid
an antibiotic that is used for controlling bacterial infections, works by binding to DNA gyrase and stopping DNA replication
38
Elongation- synthesis of primers
* DNA polymerases cannot initiate DNA synthesis on a bare template, it requires a free 3`-OH group to add a new nucleotide. * A 10-12 nucleotide long RNA primer, synthesized by an enzyme called Primase (DnaG), provides a free 3`-OH group to the DNA polymerase
39
Elongation- DNA synthesis by DNA polymerase III
* Once DNA is in a single-stranded form and a primer has been added, DNA polymerase starts synthesizing DNA. * DNA polymerase III is the main enzyme doing bulk of DNA replication. It has two catalytic activities- * 5`-3` polymerase activity- adds nucleotides to the 3` end of the growing chain. * 3`-5` exonuclease activity-removes wrong bases. * Other DNA polymerases present in bacteria are listed in a table
40
Elongation- synthesis of DNA by DNA polymerase I
DNA polymerase I with its 5`-3` exonuclease activity removes the ribonucleotides (RNA primer) and synthesizes DNA
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Elongation- DNA ligase
The breaks in the DNA chain are sealed by the enzyme DNA ligase
42
Termination
* There are different ways of termination in different types of DNAs * Replication terminates when two replication forks meet * Some DNA use specific termination sequences called Ter sites. A Tus protein binds at the ter site and causes termination of replication
43
Initiator protein
DnaA binds at oriC to initiate replication
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DNA helicase
DnaB unwinds DNA at replication fork
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Single-strand-binding (SSB) proteins
binds to ssDNA to prevent reannealing
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DNA gyrase
moves ahead of the replication fork, relieves torque
47
DNA primase
Synthesizes a short RNA primer to provide a 3'-OH group for the attachment of DNA nucleotides
48
DNA polymerase III
synthesizes DNA by extending the RNA primer. It leaves the DNA as it touches the 5`end of the previously made fragment
49
DNA Pol I
removes RNA primers and replaces them with DNA
50
DNA ligase
joins Okazaki fragments by sealing breaks in the sugar–phosphate backbone of newly synthesized DNA
51
Replicon
is an individual unit of DNA with an origin of replication
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Origin of replication
the point where replication starts
53
Eukaryotic chromosomes origins of replication
Eukaryotic chromosomes have multiple origins of replication, in contrast to bacteria that have a single origin to replicate the whole chromosome
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Modes of DNA replication
* Theta replication * Rolling circle replication * D-loop replication * Linear replication
55
Theta replication
* John Cairns in 1963 provided a visible evidence of theta replication by growing cells in radioactive growth media * DNA from cells grown in (3H) thymidine for one replication cycle were autoradiographed * In the semiconservative mode of replication, one whole strand will be radioactive (light blue) and the other strand will remain non-radioactive * Cells were grown in (3H) thymidine for another round of replication for a short time intervals and autoradiographed * these moon shaped patterns (called theta structures) of various sizes were seen * This experiment also proves that DNA replicates in a semiconservative mode
56
Rolling circle replication
* Some plasmids, bacteriophages and viral DNA replicate through unidirectional replication. * A single circular DNA produces multiple copies of the genome. * Replication starts with a nick at the origin of replication (B). * DNA polymerase binds to the nick and starts adding nucleotides at the 3` end, the old strand is displaced (C) * The polygenomic tail (D) can be cut to give unit genome length or it can be replicated to the double stranded form and then cleaved
57
D-loop replication
* Replication of mitochondrial and chloroplast DNA initiates with the formation of a D-loop (displacement loop) * One strand (The L strand) is displaced to allow synthesis of the primer on the H strand.- Primer synthesis on the L strand does not take place at this stage. * The primer is extended to replicate the H strand. * When the H strand is nearly complete then replication of the L strand initiates with the synthesis of a primer.
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Specific challenges in eukaryotic DNA replication
* DNA template is associated with histones vs no packaging proteins in prokaryotes * Genomes are larger vs much smaller size in prokaryotes * Chromosomes are linear vs circular in prokaryotes
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DNA template is associated with histones vs no packaging proteins in prokaryotes
* Eukaryotic DNA is packed with histones, so replication will require disruption of the chromatin structure and the reassembly of nucleosomes to pack newly synthesized DNA. * The disruption of the chromatin structure occurs by the replication fork. * The nucleosomes on each daughter DNA molecule have a mixture of histones from the old nucleosomes and newly synthesized proteins
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Genomes are larger vs much smaller size prokaryotes
* Eukaryotes have multiple origins of replication. It makes the process of DNA replication faster for the large eukaryotic chromosomes * ~10,000-100,000 replication origins in a human somatic cell. * A number of DNA polymerases (see Table 12.5 on the next slide) are involved in DNA synthesis. * The three polymerases alpha, delta and epsilon carry out nuclear DNA replication
61
Eukaryotic chromosomes are linear vs circular in prokaryotes
* DNA replication of the top strand is shown in B, C and D. * First the RNA primers are degraded (C). * DNA polymerase fills gaps by extending DNA synthesis. All internal gaps are filled (D). * The terminal gap can not be filled by DNA polymerase (why?), leaving tips single stranded. * It will cause shortening of the chromosome
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Enzyme telomerase
* The tips of chromosomes have specialized DNA called telomeres. * Telomeres are made of 100 to1000 repeats of a small sequence. * In humans and other mammals, the telomeres have a sequence TTAGGG which is repeated several times. * The repeats do not code for an RNA or a protein product but have an important role in DNA replication. * The repeats are added by an enzyme called telomerase, a reverse transcriptase. * The telomerase carries a small RNA molecule which acts as a template to synthesize the ‘repeat’
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'Repeats' synthesised by telomerase
two steps: (a) extension of 3` overhangs and (b) synthesis of the complementary strand.
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Telomere lengthening
Extension of the 3` overhang: * Telomerase containing an RNA component binds to the 3` overhang. * Telomerase uses RNA as a template and synthesizes DNA. (This enzyme activity is known as RNA dependent DNA polymerase or reverse transcriptase) * Once the RNA template is replicated, telomerase translocates making the template available for further DNA synthesis. * This way telomerase can extend the 3` end without the template strand.
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Synthesis of the complementary strand
Synthesis of the complementary strand: * The 5` end of the tip is synthesized by conventional replication. * As the telomerase leaves, the DNA polymerase α synthesizes an RNA primer and then DNA is synthesized. * The RNA primer is later removed leaving a small overhang at the 5` endThe 5` end of the tip is synthesized by conventional replication. As the telomerase leaves, the DNA polymerase α synthesizes an RNA primer and then DNA is synthesized. * The RNA primer is later removed leaving a small overhang at the 5` end
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Some DNA ends are replicated by using protein primers
* This is an example where a non-nucleic acid molecule primes DNA synthesis to replicate linear DNA. * Protein primers are used by several viruses ( including bacteriophage Phi 29 and mammalian viruses adenovirus and poliovirus) linear plasmids and chromosomes of Streptomyces. * A protein binds at the 3` end displacing the complementary strand. The protein has a 3` OH free for priming DNA synthesis. DNA polymerase synthesizes DNA
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