Download DNA REPLICATION Complexity of DNA

DNA REPLICATION
REPLICATE - reproduce or make an exact copy
Complexity of DNA
Complexity of DNA
- Size considerations
- Packaging (Eukaryotic DNA)
___________________________________________
Organism
kb
Length (μm)
Viruses
Polyoma or SV40
5.1
1.7
Lambda phage
48.6
17
T2 phage
166
56
Vaccinia
190
65
Bacteria
Mycoplasma
E. coli
Eukaryotes
Yeast
Drosophila
Human
760
4,000
260
1,360
13,500
165,000
2,900,000
4,600
56,000
990,000
Virtually all eukaryotic DNAs are folded and
packaged into dense, compact structures and
associated with tightly packed DNA-binding
proteins which help to organize the package
(chromatin). These structures surely are
impediments to the replication mechanism that
requires that the two parental strands be totally
separated by the end of the process.
Prokaryotes: 1 kb/sec
Euckaryotes:
Euckaryotes: 0.050.05-0.1 kb/sec
The fundamentals of DNA replication
A. DNA is replicated semisemi-conservatively in that each daughter molecule
ends up with a parental strand and a nascent strand.
DNA replication is semi-discontinuous
The fundamentals of DNA replication
So…
B. Replication is initiated at an origin of replication,
replication or multiple origins in
nuclei, and growth of each "replicon"
replicon" is usually bidirectional with both
strands being replicated simultaneously.
DNA synthesis is
semi-discontinuous
&
semi-conservative
The fundamentals of DNA replication
Most DNA polymerases have 3' to 5' exonuclease activity,
activity called
“proofreading”
proofreading”. Polymerases alpha and beta are exceptions that lack proofreading.
C. Synthesis is carried out by DNA polymerases
D. All DNA polymerases synthesize in the 5' to 3' direction (i.e., read the
template strand in the 3' to 5' direction).
5’
3’
3’
3’
5’
5’
3’
5’
*
3’
3’
5’
Two DNA polymerase (prokaryotic DNA Pol I and eukaryotic Pol ε) also have a
5' to 3' exonuclease activity that functions in repair. Both are involved in primer
removal.
5’
3’
5’
5’
xxxx
3’
3’
The fundamentals of DNA replication
E.
DNA Replication is thermodynamically favorable due to:
1.
2.
3.
4.
5’
Mechanisms
Eukaryotic and prokaryotic mechanisms are very similar.
A. Initiation at ori (origin of replication)
dNTPs ÆdNMPs + PPi
PPi Æ2Pi (loss of product)
Base stacking
Hydrogen bond formation
E.coli
•
dsDNAdsDNA-binding proteins (dnaA in E. coli) promote
unwinding at ori.
DNA polymerase - elongates the primer with deoxynucleotides.
•
ssDNA-binding proteins keep the separated
ssDNAs in an extended form.
Eukaryotic DNA polymerases:
•
Helicase uses ATP's to mechanically invade and
separate strands at the replication fork.
•
Primase synthesizes short RNA primers of 3-20
nucleotides in length.
Synthesis is in the 5' to 3' direction giving a free 3'-OH for
subsequent nucleotide extension. In prokaryotes the primase is
called “primase,” (no surprise there); but in eukaryotes, the DNA
Pol alpha is the RNA-synthesizing primase
NOTE: With the exception of Pol alpha,which is a
primase, DNA polymerases cannot initiate a new strand
de novo. They can only elongate a pre-existing primer.
DNA Pol alpha
DNA Pol beta
DNA Pol gamma
DNA Pol delta
- primes both strands.
- functions in repair of nuclear DNA.
- carries out all mitochondrial DNA synthesis.
- replication elongation of both strands (very
Processive*). This one does most of the work.
DNA Pol epsilon - involved in repair of nuclear DNA & probably
primer removal, using its 5’ to 3’ exonuclease
activity..
*Processivity is the average number if nucleotides added before the enzyme dissociates from
the DNA.
Prokaryotic DNA polymerases:
Topoisomerases
DNA Pol I - involved in repair synthesis and primer removal with
its 5’ to 3’ exonuclease activity. This was the first
DNA polymerase to be characterized.
DNA Pol II - participates in repair.
DNA Pol III - replication elongation (highly processive). This one
does most of the replicative work in bacteria.
I won the
Nobel Prize
in 1959!
My Dad won
a Nobel prize…
where’s mine?
Arthur Kornberg
DNA Pol III
Replication fork
Origin
Lagging strand
Okazaki fragments
Continuous
strand
5'
3’
3’
5’
Continuous
strand
Lagging strand
Okazaki fragments
Origin
RNA Primer
DNA Elongation
Topoisomerase II - cleaves both strands, allows unwinding of the
overwound Watson and Crick strands (an energy-requiring
process) and rejoins the ends. Uses ATP as a co-factor. A
version of this enzyme in bacteria is called "gyrase." Gyrase
makes a double strand scission, forcibly underwinds the DNA
and reseals the scission. The result is underwinding strain that
is relieved by supercoiling. The bacterial topoisomerase II is
more crucial than the topoisomerase I to the viability of the
cell.
Thomas Kornberg
DNA Pol I
Replicon
Topoisomerase I - cleaves one strand of the parental DNA beyond
forks, permits further unwinding of parental DNA, then reseals
(rejoins) the nicked ends.
Replication mechanisms (cont)
•
DNA polymerase (PolI and Polε in Prok. and Euk., respectively)
fills the gaps on lagging strands where the primers used
to be.
•
DNA ligase creates the missing phosphodiester bond
between the Okazaki fragments on the lagging strand,
using either ATP or NAD+ as a cofactor.
•
The process continues bi-directionally until both strands
are completely replicated yielding two identical daughter
molecules each having a parental strand and a nascent
strand.
Overall rates of the replication process
A.
B.
Prokaryotic - 1000 bp/fork/sec.
Eukaryotic – 100 bp/fork/sec. The rate of nuclear DNA
replication is dramatically enhanced, however, by
simultaneous initiation at multiple origins forming
multiple replicons. Each replicon expands bidirectionally
and eventually coalesces with its neighbors.
DNA Pol III acts as a Dimer with Multiple
Subunits
Two β subunits of E.coli PolIII
PolIII at the replication fork
Mechanism of PolIII action
The E. coli DNA polymerase III acts as a dimer, and each
monomer consists of multiple subunits. One monomer acts
in a continuous fashion on the leading template strand,
while the other acts simultaneously on the lagging strand
with the discontinuous formation of multiple Okazaki
fragments that are subsequently stitched together. The βsubunits form clamps that encircle the template DNA
strands, hence promoting highly processive synthesis.
DNA synthesis on the leading and lagging strands
DNA synthesis on the leading and lagging strands
DNA synthesis on the leading and lagging strands
DNA synthesis on the leading and lagging strands
DNA synthesis on the leading and lagging strands