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DNA replication
(Lecture 28,29)
1. DNA replication and the cell cycle
2. DNA is Reproduced by Semiconservative Replication
2.1 Conservation of the Original Helix
2.2 The Meselson-Stahl Experiment
2.3 Semiconservative Replication in Eukaryotes
2.4 Origins, Forks and Units of Replication
3. Enzymes involved in DNA Synthesis in Bacteria
3.1 DNA Polymerase I
3.2 Synthesis of Biologically Active DNA
3.3 DNA Polymerase II and III.
DNA is hereditary material
In 1928, Frederick Griffith showed that genetic material is a specific molecule.
Two distinguishable strains of Spreptococcus pneumoniae (a bacterium that causes pneumonia in
mammals): with smooth colonies (S) and rough colonies (R).
Cells of the smooth strain were encapsulated with a polysaccharide coat and cells of the rough strain
were not.
These alternative phenotypes were inherited
Transformation of bacteria
Griffith discovered that
(a) the S strain of the bacterium Streptococcus pneumoniae, which was protected from a mouse's
defensive system by a capsule, was pathogenic;
(b) the R strain, a mutant lacking the capsule, was nonpathogenic;
(c) heat-killed S cells were harmless; but
(d) a mixture of heat-killed S cells and live R cells caused pneumonia and death.
(e) Live S bacteria could be retrieved from the dead mice injected with the mixture.
Griffith concluded that molecules from the dead S cells had genetically transformed some of the living
R bacteria into S bacteria.
In 1944, Oswald Avery, Maclyn McCarty and Colin MacLeod discovered that the transforming agent
had to be DNA
More evidence came from studies of bacteriophages – viruses that infect bacteria
The Hershey-Chase experiment
Viral proteins, labeled with radioactive sulfur, remained outside the host cell during infection.
32P labeled viral DNA entered the bacterial cell
Additional evidence that DNA is genetic material
A eukaryotic cell doubles its DNA content prior to mitosis
During mitosis, the doubled DNA is equally divided between two daughter cells
An organism’s diploid cells have twice the DNA as its haploid gametes
In 1947, Erwin Chargaff analyzed the DNA content of different organisms:
DNA composition is species-specific
The amount and ratios of nitrogenous bases vary from one species to another
The number of adenine (A) residues approximated the number of thymines (T), same was true for
guanines (G) and cytosines (C).
The A=T and G=C equalities became known later as Chargraff’s rules.
Watson and Crick – building models to confirm to x-ray data
DNA is helix with a uniform width of 2 nm.
Purine and pyrimidine bases are stacked .34 nm apart
The helix makes one full turn every 3.4 nm along its length
There are ten layers of nitrogenous base pairs in each turn of the helix
To be consistent with a 2 nm width, a purine on one strand must pair (by hydrogen bonding) with a
pyrimidine on the other antiparallel helix
Base structure dictates which pair of bases can hydrogen bonds
Consistence of base-pairing rule
It explains Chargraff’s rules
It suggests the general mechanism of replication
It dictates the combination of complementary base pairs and places no restriction on the linear
sequence along the length of a DNA strand.
Watson and Crick’s model of replication
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
Conservative replication: complementary chains are synthesized as for semiconservative, but the two
newly created strands then come together and the parental strand reassociate.
The original helix is “conserved”
Dispersive replication: the parental strands cleave during replication and the DNA is then dispersed
into two new double-helices.
Each strand consists of both old and new DNA – original helix is not conserved.
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.
Semiconservative Replication in Eukaryotes
1957 – J.H. Taylor, P. Woods, W. Hughes presented the evidence
occurs in eukaryotes.
that semiconservative replication
Origins, Forks and Units of Replication
DNA replication is initiated at origin.
At the point of origin the strands of the helix unwound, creating a replication fork.
The unit of DNA in which an individual act of replication occurs is called the replicon.
Beside an origin, a replicon can contain a terminus – a place where replication ends
Is the a single origin or replication can start at several places?
Does replication proceed in one direction (unidirectional) or in both (bidirectional)?
Replication starts at several places and is bidirectional.
The two strands of DNA are antiparallel
The problem of antiparallel DNA strands is solved by the continuous synthesis of one strand (leading
strand) and discontinuous synthesis of the complementary strand (lagging strand)
Synthesis of leading and lagging strands
Leading strand – the DNA strand which is synthesized as a single polymer in the 5’ ◊ 3’ direction
towards the replication fork.
Lagging strand – the DNA strand that is discontinuously synthesized against the overall direction of
replication.
Priming DNA synthesis
Primer – short RNA segment that is complementary to a DNA segment and that is necessary to begin
DNA replication.
Steps of DNA replication
1. Double helix unwinds, providing single-stranded DNA templates. This is done with the help of
helicases and single-strand binding proteins.
2. Synthesis of leading strand.
Priming (done with the help of primase)
Elongation (DNA polymerase)
Replacement of RNA primer by DNA (DNA polymerase)
3. Synthesis of lagging strand
Priming for Okazaki fragment (primase)
Elongation of fragment (DNA polymerase)
Replacement of RNA primer (DNA polymerase)
Joining of fragments (DNA ligase)
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 (UV-sensitive).
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
Properties of Three Bacterial DNA Polymerases
I
II
Initiation of chain synthesis
-
III
-
5’-3’ polymerization
+
+
+
3’-5’ exonuclease activity
+
+
+
5’-3’ exonuclease activity
+
-
-
?
15
Molecules of polymerase/cell
400
Role of the 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.
Features of eukaryotic replication
1. Eukaryotes have 100-1000 times larger chromosomes
2. Rates of DNA replication in eukaryotes is 1000 fold slower (in E. coli chromosome replicates 40
min)
3. Tight control over replication (S phase lasts 9 hrs)
4. Multiple origins of replications, autonomously replicating sequences (ARS).
Summary
1. DNA replication is semiconservative
2. Ones replication has started it continues until the entire genome has been duplicated
3. It starts at origin. An origin “fires” ones and only ones during the cell cycle.
4. It is bidirectional
5. At the place of the replication start (origin) helix unwinds and creates two replicational forks
6. Enzymes that synthesise new strands are called polymerases and have complex structure and
function including proofreading activity.
Reading
CH. 16 (293-308)