DNA repair

DNA repair
Damaged DNA must be repaired
If the damage is passed on to subsequent
generations, then we use the evolutionary
term - mutation. It must take place in the
germ cells - the gametes - eggs and sperm
If damage is to somatic cells (all other cells
of the body bar germ cells) then just that
one individual is affected.
Damage from where?
Consequences of DNA replication errors
Chemical agents acting on the DNA
UV light imparting energy into DNA
molecule
Spontaneous changes to the DNA

Why repair DNA?
DNA pol does a great job, but not good enough
Introduces errors in about 1 in 10E7 nucleotides
added, which it does not correct
Other mechanisms exist (as we will see) to
correct many of the errors left by the replication
system
Most mistakes and damage corrected (99% -
leaving just a few - only 1 in 10E9 errors are left)
Mutations are permanent changes left in the
DNA

Why repair DNA?
Repair of non-replication related damage to the
DNA must also be a priority for the cell.
These defects also will prevent translation and
duplication of the DNA
Cell will die.
Again, any errors or changes to the DNA
become Mutations - which are permanent
changes left in the DNA

Sickle Cell Disease
This is a very good illustration of the devastating
effects of even tiny changes to the DNA
Red Blood Cells
Hemoglobin -
Has a large protein component
2 beta globin chains
A single base change -substitution causes the disease

06_19_sickle_cell.jpg
Spontaneous Mutations
Involves thermal energy
Due to random molecular collisions
between molecules and DNA in the cell
Cannot be prevented
Parts of the DNA molecule are stripped off
and alterations introduced
Many outcomes

Direct DNA Damage
Some agents damage DNA directly
Chemicals and light
Chemicals - alkylating agents
Methy and ethyl groups added to DNA bases
This type of damage can be repaired by direct
reversal involving special enzymes
They remove the offending atoms and restore the base

DNA Damage
Just a few types of damage is repaired via
simple reversal of the chemical change -
UV light induced dimers
Methylation of bases
Ethylation of bases
Large chemical groups added to the DNA
Most other damage require other
systems
06_24_radiation.jpg
Random photons of ultraviolet (UV) light induce aberrant bonding
between neighbouring pyrimidines (thymine & cytosine) bases on
the same strand of DNA. The will prevent the replication machine
from duplicating the DNA. The cell will die!
This type of defect can be readily reversed by a process called
photoreactivation. Visible light energy is used to reverse the defect (in
bacteria, yeasts, protists, some plants, and some animals but NOT in
humans)
Other forms of DNA damage
Deamination - An amino group of Cytosine
is removed and the base becomes Uracil
Deamination - An amino group of Adenine
is removed and the base becomes
Hypoxanthine
Deamination - An amino group of Guanine
is removed and the base becomes
Hypoxanthine
And
Depurination - the base is simply ripped
out of the DNA molecule leaving a gap
(like a missing tooth)
06_23_Depurination.jpg
Molecular level view-
Remember these are random events
06_25_mutations.jpg
DNA level view of the same two events as last slide
Which is which?
The cell has a big problem to overcome
How does it tell which strand carried the
correct information?

We think we know
06_21_Errors corrected.jpg
The cell has to pick the right strand to fix or else
06_22_DNA mismatch.jpg
The cell has a mechanism of identifying new strand synthesis by
leaving nicks that DNA. There are enzymes which scan these new regions
looking for errors
Correction mechanisms
Direct reversal of damage -
Photoreactivation (bacteria, yeast,
some vertebrates - not humans) Two
thymines connected together by UV
light.
Excision Repair - removal of defective
DNA. There are three distinct types
1) base-excision repair
2) nucleotide-excision repair
3) mismatch repair
Direct reversal of DNA damage
Photoreactivation
Direct reversal of DNA damage
The removal of the methyl group
Excision Repair
1. base-excision repair
Presence of the Uracil in DNA is a great
example of this type
Special enzymes replace just the defective
base
1 snip out the defective base
2 cut the DNA strand
3 Add fresh nucleotide
4 Ligate gap
DNA Repair: Base
Excision Repair (BER)
Steps in BER process in
eukaryotes
Initiated by a DNA
glycosylase that
recognizes alteration
DNA glycosylase removes
the base (not the entire
nucleotide)



base-excision repair
DNA Repair: Base Excision
Repair (BER)
Steps in BER process in
eukaryotes
The "beheaded" deoxyribose
phosphate is removed by (AP)
endonuclease & DNA polymerase
1. The AP endonuclease cleaves
the DNA backbone
2. Polymerase removes the
sugar-phosphate remnant that had
been attached to the excised base
3. Gap filled by DNA polymerase
4. Strand is sealed by DNA ligase



base-excision repair
2. Nucleotide excision repair
(NER)
Recognizes bulky lesions that block DNA
replication (i. e. lesions produced by
carcinogens)--example, UV pyrimidine
photodimers
Common distortion in helix
Incision on both sides of lesion
Short patch of DNA excised, repaired by
repolymerization and ligation
In E. coli, mediated by UvrABCD
Many more proteins involved in eukaryotes
Can be coupled to transcription (TCR,
transcription coupled repair)
Defects in NER underlie Xeroderma
pigmentosum
Xeroderma pigmentosum
Autosomal recessive mutations in several complementation
groups
Extreme sensitivity to sunlight
Predisposition to skin cancer (mean age of skin cancer = 8
yrs vs. 60 for normal population)
Recognition and binding
UvrA acts as classical
molecular matchmaker
Incision
Nicks delivered 3
and 5 to lesion by
UvrBC
Excision and repair
Short fragment
released by
helicase action


S. cerevisiae protein Human protein Probable function
Rad14 XPA Binds damaged DNA after XPC or RNA pol II
Rpa1,2,3 RPAp70,p32,p14 Stabilizes open complex (with Rad14/XPA); positions
nucleases
Rad4 XPC Works with hHR23B; binds damaged DNA;
recruits other NER proteins
Rad23 hHR23B Cooperates with XPC (see above); contains ubiquitin
domain; interacts with proteasome and XPC
Ssl2 (Rad25) XPB 3' to 5' helicase
Tfb1 p62 ?
Tfb2 p52 ?
Ssl1 p44 DNA binding?
Tfb4 p34 DNA binding?
Rad3 XPD 5' to 3' helicase
Tfb3/Rig2 MAT1 CDK assembly factor
Kin28 Cdk7 CDK; C-terminal domain kinase; CAK
Ccl1 CycH Cyclin
Rad2 XPG Endonuclease (3' incision); stabilizes full open complex
Rad1 XPF Part of endonuclease (5' incision)
Rad10 ERCC1 Part of endonuclease (5' incision)
Proteins Required for Eukaryotic Nucleotide Excision Repair
Human NER
Rad1/10
Rad2 in S. cerevisiae
3. Mismatch repair
Special enzymes scan the DNA for bulky
alterations in the DNA double helix
These are normally caused by mismatched
bases
AG
AC
CT
These are excised and the DNA repaired
06_26_three steps.jpg
Basic mechanism is
the same for all
three types
1) Remove damaged
region
2) Resynthesis DNA
3) Ligate
mismatch repair

Evolution acts on mutations
If we did not have mutation then we would
all be the same!
Any changes in the environment would be
deleterious to all members of the
population equally
= There would be no evolution!!!!
But mutation does exist and it is supported
by comparison of related organisms
06_27_humans_whales.jpg
THANK YOU