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.jpgRandom 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.jpgMolecular level view-Remember these are random events

06_25_mutations.jpgDNA 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.jpgThe 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 regionslooking 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 β removes the sugar-phosphate remnant that had been attached to the excised base

3. Gap filled by DNA polymerase β 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

nucleasesRad4  XPC  Works with hHR23B; binds damaged DNA;

recruits other NER proteinsRad23  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.jpgBasic mechanism is the same for all three types

1) Remove damaged region

2) Resynthesis DNA3) 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