Download - Dna Repair
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
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