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

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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

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!

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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)

Molecular level viewRemember these are random events

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DNA level view of the same two events as last slide

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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

The cell has to pick the right strand to fix or else

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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

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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

base-excision repair
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

 Recognizes bulky lesions that block DNA

2. Nucleotide excision repair (NER)

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)

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

Incision

Excision and repair

UvrA acts as classical molecular matchmaker

Nicks delivered 3 and 5 to lesion by UvrBC

Short fragment released by helicase action

Proteins Required for Eukaryotic Nucleotide Excision Repair

S. cerevisiae protein Human protein Rad14 XPA Rpa1,2,3 RPAp70,p32,p14 Rad4 Rad23 Ssl2 (Rad25) Tfb1 Tfb2 Ssl1 Tfb4 Rad3 Tfb3/Rig2 Kin28 Ccl1 Rad2 Rad1 Rad10 XPC hHR23B XPB p62 p52 p44 p34 XPD MAT1 Cdk7 CycH XPG XPF ERCC1 Probable function Binds damaged DNA after XPC or RNA pol II Stabilizes open complex (with Rad14/XPA); positions nucleases Works with hHR23B; binds damaged DNA; recruits other NER proteins Cooperates with XPC (see above); contains ubiquitin domain; interacts with proteasome and XPC 3' to 5' helicase ? ? DNA binding? DNA binding? 5' to 3' helicase CDK assembly factor CDK; C-terminal domain kinase; CAK Cyclin Endonuclease (3' incision); stabilizes full open complex Part of endonuclease (5' incision) Part of endonuclease (5' incision)

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

Basic mechanism is the same for all three types 1) Remove damaged region 2) Resynthesis DNA 3) Ligate

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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

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THANK YOU