(Genotype)
Replication
(Chemistry, Phenotype)
Replication
(Chemistry, Phenotype)
DNA Replication
Action of DNA polymerase. DNA polymerases
assemble incoming deoxynucleoside triphosphates
on single-stranded DNA templates such that the
growing strand is elongated in its 5’ to 3’ direction.
Priming of DNA synthesis by short
RNA segments.
DNA Polymerase
DNA polymerase activity
3’-5’ exonuclease
5’-3’ exonuclease
3’-5’ exonuclease cleaves
misincorporated
nucleotides
5’-3’ exonuclease
Cleaves RNA primers from
growing strands
1) Helicase: separate DNA strands
2) DNA primase: RNA polymerase that lays down the
primer
3) DNA polymerase replicates DNA
4) b clamp increases processivity
5) DNA gyrase/topoisomerase: prevents supercoiling
Helicase and gyrase/topoisomerase
E. coli DNA replicated at 1000 nucleotides/s
1300 µM DNA would flail around at 100 revolutions/s
Since it is circular it instead supercoils
Accumulates +100 supercoils/s these need to be alleviated
DNA is naturally negatively supercoiled and this promotes
unwinding
However, GyrA, type II topoisomerase further helps by introducing
negative supercoiling
Proofreading
E. coli polymerase makes mistakes every 109 -1010
nucleotides added
Given that the genome is 4.6x106 bp this means
1 error per 1000 to 10000 replications
Proofreading requires
Proper base pairing
Even a properly inserted nucleotide is removed a
significant amount of the time just to be sure….
Many types of DNA polymerase in E. coli
Pol I: DNA repair, excision of RNA primers
Pol II: DNA repair
Pol III: main DNA replicase
Pol IV and V?
Eukaryotic DNA polymerases
Pol : a primase
Pol : main polymerase
Pol : mitochondrion
Pols b: DNA repair
The reactions catalyzed by reverse
transcriptase.
NMR structure of the telomeric
oligonucleotide d(GGGGTTTTGGGG).
Errors do occur
Point mutations
Insertions
Deletions
Rearrangements
Spontaneous mutation rate = 10-10 to 10-12/cell
division in bacteria
1 nucleotide change/per 1000 takes 200,000 yrs. in humans
Types and sites of chemical damage to
which DNA is normally susceptible in
vivo. Red, oxidation; blue, hydrolysis;
green, methylation.
Mispairing of A with C or G with T: Tautomerization
H
O
O
H
N
N
A
N
H
O
N
H
N
N
G
T
H
N
N
A
N
N
H
N
H2N
N
N
H
N
C*
N
N
O
C
N
O
T*
N
N
H
O
H
N
H
H
N
N
N
N
O
Hydrolysis
Need to get rid of uracil in DNA
Hydrolysis
Depurination
occurs 6000
times per day
in a
mammalian
cell
Methylation
Oxidation
O
N
O
N
¥OH
NH
NH
HO
N
H
N
H
NH2
N
H
N
NH2
Guanosine
O
O
H
N
N
HO
HO
H
NH
NH
N
H
N
NH2
N
H
N
NH2
Hydroxoguanosine
Oxidation
P
base
O
O
H
P
¥OH
H
base
O
O
H
H
H
H
H
O
P
P
P
O
O
O
H
O
H
H
O
CH3
O
H
H
H
H
H
H
O
P
Strand Breaks
H
Deletions due to strand breaks
Insertions and deletions
Caused by repetitive sequences
and intercalators
abasic sites
single strand breaks
Triplet repeat expansion/contraction
CAG or CGG repeats (polyGln)
Triplet repeat expansion/contraction
CAG or CGG repeats (polyGln)
Deletions
Six main types of repair mechanism
Direct repair
Mismatch repair
Base Excision Repair
Nucleotide Excision Repair
SOS Repair
Homologous recombination
Direct repair: photolyase
Direct repair: O6-methyl guanosine demethylase
Mismatch repair
Points out into major groove
H3C
N
N
N
H
H
N
N
Base Excision Repair
Nick translation by DNA pol I
Nucleotide Excision Repair
SOS Repair/Translesion synthesis/Error prone polymerase
When you hit a lesion DNA pol III falls off
It is replaced by pols without proofreading
this causes the polymerase to pass over
the lesion without changing it
This, of course,
introduces errors but it is
better than no replication
at all
So stress induces deliberate mutation!
Why?
To enhance evolvability, perhaps?
One of the best ways to repair DNA is to use
homologous recombination: sharing
information between homologous
chromosomes
Homologous end-joining
Gross
Rearrangements
Transposon and transposable elements
DNA only transposons
Retroviral like retrotransposons
Nonretroviral retrotransposons
Transposons are ‘jumping genes’ that
make use of recombination to jump around
Chromosomal rearrangement via recombination. (a) The
inversion of a DNA segment between two identical
transposons with inverted orientations.
Chromosomal rearrangement via recombination. (b) The
deletion of a DNA segment between two identical
transposons with the same orientation.