repair.ppt

DNA damage and 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…

General strategies for DNA repair

Direct Reversal of DNA damage: • PhotolyasesPhotolyases• AlkyltransferasesAlkyltransferases

• Nucleotide Excision Repair (NER)Nucleotide Excision Repair (NER)

Double-Strand BreakRepair:

• Non-homologous end-joining (NHEJ)Non-homologous end-joining (NHEJ)• Homologous recombination (HEJ)Homologous recombination (HEJ)

• Mismatch repair (MMR)Mismatch repair (MMR)

Excision-Repair: • Base Excision RepairBase Excision Repair

Error-prone Repair: • Trans-lesion replication (TLS) (SOS)Trans-lesion replication (TLS) (SOS)

• Transcription-coupled Repair (NER)Transcription-coupled Repair (NER)

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

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•2) nucleotide-excision•3) mismatch repair

base-excision

•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

nucleotide-excision

•Same as previous except that•It recognizes more varieties of damage•Remove larger segments of DNA (10 -100s of bases)

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

Consider…

• Sunlight - sunbathing or daily exposure• Impact of ozone depletion• Impact on different skin tones

• Environmental degradation

DNA repair diseases

Defects in genes encoding proteins involved in mismatch repair, nucleotide-excision repair, and recombinational repair can cause cancer

Nucleotide-excision repairsole repair pathway for pyrimidine dimers

genetic defect causes XP, xeroderma pigmentosa, these individuals are extremely sensitive to sunlight and quickly develop sunlight-induced skin cancer

Mismatch repairHereditary nonpolyposis colon cancer (HNPCC) linked to defects in these genes

Defects in human MutL homolog, human MutS homolog

Recombinational repairRecombination - linear sequence of DNA altered by cleavage and rejoining of chromosome (involves RecA protein)

Repair of this type sometimes needed to reconstruct replication fork

Human breast cancer genes (BRCA1 and BRCA2) produce proteins that interact with the human homolog of RecA, therefore these are linked to recombination repair

10% of breast cancers have defects in BRCA1 or BRCA2Women with defects in these genes have a >80% chance of developing breast cancer

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…