BIOL 2010 - DNA damage and repair

Types of DNA damage

• abnormal base pairs

• chemical adducts —> stop replication fork/DNA pol

• incorrect bps

• double stranded break —> fragments may be lost

• single stranded break

• abasic site —> nucleotide still there but no base

• thymine dimers —> DNA pol cant get past

• DNA cross links —> covalent bonds instead of H bonds

• nucleotide insertions/deletions

spontaneous vs induced causes of DNA damage

SPONTANEOUS

1. replication errors

2. tautomerisation —> the addition of a proton at one molecular site and the removal of a proton at another

3. deamination

4. depurination

INDUCED

1. intercalating agents

2. base analogues

3. deaminating agents

4. oxidising agents

5. radiation, UV

Deamination/depurination

DEAMINATION

• deamination of CAG can be repaired but deamination of T cant be repaired as its not considered wrong

• deaminating agents will work much faster than spontaneous deamination

• Guanine is much more prone to mutation than other bases as it has more sites of attack

DEPURINATION

• depurination will not result in mutation until it becomes a template strand

• 4x more likely in ssDNA

AMES test

• bacteria are modified so that they require histidine so only grow if histidine is present

• can test if something is a mutagen by testing if the bacteria can grow on an agar plate without histidine

• the mutagen will cause a mutation in the gene which allows the bacteria to produce histidine

intercalating agents

• fit between the tacked bases of DNA and change the width of the DNA strand

• leads to distortion of the helix and local unwinding

• can lead to slippage or improper base pairing which can cause frameshift mutations

• E.g ethidium bromide

base analogues

• molecules which resemble nucleotides and insert themselves into DNA but have altered pairing properties

• E.g 5 bromouracil

oxidising agents

• highly reactive oxygen species derived from by-products of cellular reactions

• can cause strand breaking or cross links in the DNA

• E.g hydroxyl radicals

radiation/UV

• radiation can induce chemical changes in the DNA which alter its structure

• radiation can interact with surrounding molecules to produce reactive species which can interact with the DNA

• UV causes the formation of thymine dimers - its not a mutation but clogs up the machinery for replication

DNA repair mechanism

2 broad repair mechanisms :

• strip out and resynthesise

• direct repair to DNA

double stranded breaks

is the most serious form of DNA damage and has two repair methods:

HOMOLOGOUR RECOMBINATION

• fragments of damaged chromosome can be aligned and cross over with its homologue

• forms two holliday junctions (one for each type of the break)

• DNA synthesis and ligation correct the break

NON HOMOLOGOUR END JOINING

• Kn proteins bind the two broken ends of the duplex fragments and then to eachother

• DNA ligase IV recruited to the join

• Process is not precise - may have overhanging ss

• ligates both strands at once but some loss of nt

• risk of deletion mutation

• risk is that joins any strand of DNA —> if theres multiple breaks chromosome fixed in wrong order

p53 tumor supressor

• detects damaged DNA

• arrests the cell cycle —> gives DNA the time to repair before S phase

• activates DNA repair

• if damage is too severe activates apoptosis or sinescence

• most tumprs have mutated p53

4 types of sequence repair

1. direct repair

2. base excision repair

3. nucleotide excision repair

4. mismatch repair

1. DIRECT REPAIR

• damage is modified for normal bases

• can convert back to normal nucleotide

• E.g: methylation, removal of thymine dimers

• simple correction

DEMETHYLATION

• methyltransferase enzyme

• transfers methyl group onto itself

• sacrificial —> permenant modification of enzyme

THYMINE DIMERS

• corrected with light and enzymes

• DNA photolyase absorbs blue light and breaks T-T internucleotide bonds using FADH —> 2xTs restored

• instant repair

• mammals cant do this, must use other repair mechanisms

4 types of sequence repair

1. direct repair

2. base excision repair

3. nucleotide excision repair

4. mismatch repair

2. BASE EXCISION REPAIR

• removal and replacement of bases

• >6 DNA glycosylases which recognise abnormal bases and cleave them from the deoxyribose creating an abasic site

• can deal with:

  • deaminated A/C methyl C

  • oxidised

  • alkylated

  • open ring

  • double bond loss

• glycosylases flip out bases for closer inspection

• if the base is incorrect, cleaves the base but the enzyme remains attached to label it for the next stage

• This is done UDGase - the same enzyme which recognises the U in the lagging strand

• this makes it difficult to distinguish U and T but the steric clash of the methyl group on the T is enough to distinguish it as correct

when Us are present in DNA

• U suggests a mutation

• base is removed by glycosylase —> baseless nt

• separate protein is required for baseless nucleotides

• AP (apyrimidinic) endonuclease will close the phosphodiester backbone - this will also work in deourination

• in bacteria pol 1 nick translation restores T and DNA ligase seals the nick

• in eukaryotes pol beta does this

4 types of sequence repair

1. direct repair

2. base excision repair

3. nucleotide excision repair

4. mismatch repair

3. NT EXCISION REPAIR

• removal of a ss sequence of DNA

• triggered by distorted DNA

• can correct any kind of damage

• mammals use this to remove thymine dimers (prokaryotes will use DNA photolyase)

• UVr ABC exinuclease in E.coli removes sections of DNA

  • UVrB heterodimer scans DNA and binds distortions

  • once bound the 2x UVrA proteins lost and UVrC is recruited to the site

  • UVrB and UVrC slide away from each other (on the undamaged strand) and cut either side of the damaged section

  • specialised helicase UVrD then knocks off the fragment

4 types of sequence repair

1. direct repair

2. base excision repair

3. nucleotide excision repair

4. mismatch repair

4. MISMATCH REPAIR

• detects incorrect combo of bases

• mismatch distorts the helix which is detected by proteins

• the incorrect base is then removed

• the most newly synthesised DNA will be corrected as its more likely to be mutated

• hemimethylated DNA provides info on which is the most newly synthesised strand

IN PROKARYOTES

1. MutH binds to unmethylated daughter strand at the origin

2. MutS binds to distorted region

3. Mut L binds to Mut S and brings it to Mut H

4. Mut H cleaves the daughter strand at the origin and removes all of the DNA up to the mistake

5. UVrD removes the daughter strand a little bit past the distortion

6. pol 3 will then fill in the missing nts and DNA ligase will fill the nick

IN EUKARYOTES

• we have several homologues

  • MutL 1-5

  • Mut S 1- 6

• no homologues of Mut H as out DNA doesn’t use hemi methylation

multipronged approach - specific G mutation

• 8 oxo G is a specific G mutation

• Mut T recognises 8 oxo GTP and hydrolyses it so it can repair mutation before it even ends up in DNA

• Mut M recognises * oxo G in DNA and removes it via BER —> leaves an abasic site

• Mut Y recognises 8 oxo G opposite an A in DNA and removes the V via BER

• Gs get damaged easily so specific repair mechanism for mutated Gs

error rate of polymerases

• 10^-5

• with proofreading = 10^-7

• with the repair mechanism = 10^-10

translesion synthesis

• creates a problem when DNA replication is stopped due to incorrect or distorted nucleotides which might cause mutation

• some polymerases add nts where processive, proofreading polymerases cant

• after, the mutations can be corrected

• pol IV and pol V are translesion synthesis pols which are not too fussy about matching nts —> bypass a blockage so replication is finished

xeroderma pigmentosum

• cant repair UV damage

• skin very sensitive to UV light —> 1000 fold increased risk of skin cancer

• due to inherited defects in one of 8 distinct genes responsible for components of the NER complex

• system defective everywhere but only skin exposed to UV light so defects only shown in skin

HNPCC (colon cancer)

• mutation in homologues of mismatch repair (Mut S/Mut L)

• accumulation of mutations in genome which lead to colon cancer