Lecture 4 - DNA Repair

DNA errors can be _____ or _____ and can be introduced by several factors.

induced; spontaneous

can be induced by:

• DNA replication

• free radicals

• ionizing radiation

induced mutations occur due to a physical or chemical external agent and spontaneous mutations occur because of natural processes in cells

Mutations are not always bad

some have consequences like being deleterious or advantageous

major source of genetic variation which drives evolutionary change to allow organisms to respond to changing environments

hotspots in chromosomes have more mutations than other areas

Point Mutations

single nucleotide change

• transition mutation: pyrimidine replaced by pyrimidine or purine for purine

• transversion mutation: purine is substituted for pyrimidine (A/G ←→ T/C)

protein coding genes with point mutations can be classified as:

• silent/synonymous: nucleotide substitution that doesn’t change the amino acid encoded

• missense/non-synonymous: nucleotide substitution that results in change of amino acid encoded

• frameshift: insertion or deletion of nucleotide(s) that alters reading frame

• nonsense: nucleotide substitutions that results in creation of stop codon and makes a truncated protein

Phenotypic Consequences of Point Mutations

consequences are based on where the mutation happens in the cell

1. non-coding RNA: may alter structure and function

2. non-coding gene: affect gene regulation and splicing

3. protein coding gene: alters DNA sequence but specific effect varies

Trinucleotide Repeats

type of microsatellite

increases genetic instability in some areas of the genome since they can adopt a variety of secondary structures

repeats can expand or contract from slippage of the newly made strand on the original strand during DNA synthesis so that misaligned repeats pair up

considered a dynamic mutation because the number of triplets in the disease gene continues to increase as the disease gene is inherited

Consequences of Trinucleotide Expansions

inverted repeats → cruciform structure

repeat with mirror symmetry → triplex DNA

G-rich sequences → G-quadruplex

3 Classes of DNA Damage

• single base changes

• structural distortion

• DNA backbone damage

Single Base Changes

conversion: mutagen changes the base on the nucleotide in a DNA sequence

• generally causes minor structural distortion so might not block replication or transcription

• change base-pairing can result in transition or transversion after replication

Intrinsic and Extrinsic Agents

extrinsic agents: X-rays (oxidiser)

intrinsic agents: free radicals from metabolic processes (oxidisers), replication mistakes

DNA Backbone Damage

structural changes in helix impede replication or transcription

• may happen as a consequence of pyrimidine dimers generated by UV light where adjacent pyrimidines on the same strand are covalently linked to each other preventing their base pairing with purines in the complementary strand

• intercalating agents change the base stacking interactions

• base analogs → different base pairing interactions that affect the shape of DNA

• structural changes may also be caused by DNA adducts when DNA is chemically bonded to a cancer-causing substance

• other bulk may be introduced by covalently attaching proteins to nitrogenous bases of DNA (disrupts helical structure)

Abasic Sites

most common DNA lesions from cleavage of the N-glycosidic bond between the nitrogenous base and deoxyribose sugar, leaving an intact phosphodiester bond but no base

apurinic or apyrimidinic

Important DNA Polymerases

there are 17 in mammalian cells - 4 replicative and 13 repair

• pol alpha: primes DNA synthesis during replication and repair

• pol delta: replication of lagging strand during replication and repair

• pol epsilon: replication of LEADING strand during replication and repair

• pol gamma: mitochondrial DNA replication and repair

repair polymerases respond to damage by:

1. bypassing damage

2. reversing damage

3. removing damage

Damage Bypass

replication forks can encounter structural problems that impair the ability of the fork to progress:

• DNA lesions (T-T dimers)

• non-B DNA (triplex, slipped DNA, cruciform, G-quad)

DNA polymerases replicate through lesion by inserting an incorrect base or skipping the base → translesion synthesis

Translesion Synthesis

DNA replication factories (clusters of many replication forks in discrete subnuclear compartments) let polymerase read through the error

• gap filling: replication machinery leaves ssDNA opposite the lesion to be filled by a translesion polymerase later

• polymerase switching: replicative polymerase is displaced at the lesion and PCNA is loaded with a translesion polymerase with the aid of Rev1

how the lesion is filled is dependent on which polymerase is recruited - in prokaryotes pol IV and V are only part of SOS response as response to environmental stress or chemical mutagens

high fidelity in eukaryotes is achieved by pol eta

Damage Reversal

done by a distinct set of repair enzymes

in proks and some euks, Pyrimidine-pyrimidine dimers caused by UV light are directly repaired by photoreactivation

in proks and euks, reversal of base methylation (O6-methylguanine) is done by methyltransferases

In multicellular eukaryotes, DNA-protein crosslinks are reversed by _____.

SPTRN

Photoreactivation

damage and agent: UV light damage results in the formation of pyrimidine-pyrimidine dimers

repair:

• DNA photolyases use visible light (near-UV to blue or 300-500nm) to break the covalent bonds holding the adjacent pyrimidines together

• takes 2 co-factors: FADH for electron transfer and a chromophore for pigment

this mechanism is NOT present in placental mammals

Which mechanism for damage reversal is not present in placental mammals?

photoreactivation

Demethylation of O6-methylguanine

damage and agent: O6-methylation of guanine by alkylating agents

repair:

• o6-methylguanine-DNA-methyltransferse MGMT binds DNA in minor groove, widens it, and bends the DNA to flip out the damaged bases in the major groove

• active site of methyltransferase contains a cysteine residue that accepts the methyl group from the guanine (removes group from guanine)

• terminal reaction (kills enzyme after protein accept methyl)

Removal of DNA Protein Crosslinks

damage and agent: induced when proteins become and remain covalently attached to the nitrogenous bases, sugar, or broke phosphodiester bond on the DNA backbone

repair:

• SPRTN associates with replication machinery to degrade proteins associated with DNA and associated conformational change are essential for SPRTN activation

  • DNA-dependent metalloprotease that chews up any protein associated with DNA so must be regulated

• regulated by ubiquitin

  • unubiquitinated = on

Damage Removal

simple overview:

1. DNA repair machinery gains access to the DNA (chromatin remodelling, histone ejection)

2. DNA repair machinery removes the damage

3. gap refilled

4. histones redeposited to reform nucleosome

Types of Single Base Pair Changes

• mismatch repair

• base excision repair

Base Excision Repair

initiated by DNA glysolyases, leaves an abasic site when damaged base is excised → no base is available for pairing even though phosphodiester bond still exists

• shortpatch repair for single nucleotide

• long patch repair for 2-10 nucleotides

Shortpatch Repair

glycosylase-associated beta-lyase and apurinic/apyrimidinic endonuclease (APE1) make nicks 3’ and 5’ to the abasic site, respectively

pol-beta replaces missing nucleotide and ligase 3-XRCC1 complex seals the gaps in the sugar-phosphate backbone

Long Patch Repair

apurinic/apyrimidinic endonuclease (APE1) makes a 5’ nick to the abasic site in DNA → pol delta or epsilon (based on leading or lagging strand) and PCNA displace the strand 3’ to the nick to make a flap of 2-10 nucleotides

flap is cut at the junction of the single-to-double strand transition by flap endonuclease 1 (FEN-1)

synthesized fill in is ligated by DNA ligase I

Mismatch Repair

requires strand identification to see which strand is correct - unclear in eukaryotes but in E. coli can differentiate by presence of methylated GATC, which is the strand that gets nicked

once mismatch is recognized, more ATP-bound-MSH sliding clamps are leaded (slows down replication at mismatch and recruits proteins for repair)

DNA is then excised either 5’ or 3’ from the mismatch

5’ Mismatch Repair

in 5’ excision ATP-bound MSH sliding clamp holds EXO1 on the DNA at the 5’ strand nick and helps it move from 5’-3’

• EXO1 can then digest the strand of the DNA that contains the damage

  • continues for up to several thousand nucleotides

• as ssDNA is generated it is coated by RPA

• EXO1 can associate with different ATP-bound MSH on the DNA to digest the mismatched strand

3’ Mismatch Repair

PCNA is bound to 3’ nick

• ATP bound MSH sliding clamp associates with MLH/PMS and this complex diffuses along the DNA toward PCNA

• interaction between MSH-MLH/PMS and PCNA activates the endonuclease activity of MLH/PMS

• active MLH/PMS endonuclease introduces multiple single strand breaks in the 5’ direction from 3’ end

• these nicks act as substrates for the EXO1 5’ exonuclease → repeated until the mismatch is release and no more MSH sliding clamps are loaded

• once mismatched strand is excised the new DNA can be made by pol-delta and its associated factors

• ligase I seals the nick by forming the last phosphodiester bond

Repair Proteins in Damage Bypass

DNA polymerases IV and V in e. coli

DNA polymerase eta in humans

Repaid Proteins in Damage Reversal

DNA photolyase for photoreactivation due to pyrimidine dimers

methyltransferase for removal of methyl groups due to O6-methylguanine

SPRTN protease for removal of DNA-protein crosslinks due to DNA-protein crosslinks

Repair Proteins in Damage Removal

DNA glysosylases for base excision repair due to damaged base

MutSa, MutLa and EXO1 for nucleotide excision repair due to replication errors

XPA, XPB, XPC, XPD, ERCC1/XPF, XPG for nucleotide excision repair due to pyrimidine dimer bulky adduct on base

MRN complex, Rad51, Rad 52, BRCA1, BRCA2, XRCC3 in humans for homologous recombination 

• Ku proteins, Artemis/DNA-PKCS, XRCC4 in humans for nonhomologous end joining for double strand break repair due to double stranded breaks

DNA Repair Summary

Nucleotide Excision Repair

pathway that DNA causing structural distortion (like pyrimidine dimers) are recognized by cooperative binding of RPA, XPA, XPC, TFIIH complex to facilitate excision of damaged base and filling in of the gap and ligation by pol and ligase

deficiency in NER → xeroderma pigmentosum, trichothiodystrophy, Cockayne syndrome

Responses to Pyrimidine-Pyrimidine Dimers

• bypass: replicated/synthesized through by pols, generally error prone unless it’s pol eta

• reversal: photoreactivation by DNA photolyase but not in placental mammals

• removal: NER (highly conserved) - XP type of proteins

Xeroderma Pigmentosum (XP)

unusually sensitive to UV light and not able to efficiently repair UV damage - rare autosomal recessive disorder

• 7 of the genes associated with Xeroderma pigmentosum are involved in NER (XPA, XPB, XPC, XPD, XPE, XPF, XPG)

• 8th gene encodes for DNA pol eta

Nucleotide Excision Repair Pathway

mutations in this - XP

key players are: RPA, XPA, XPC, TFIIH, XPG, XPF/ERCC

NER is coupled to transcription and thus NER pathway repairs lesions by:

• global genome repair: NER pathway responsible for recognizing DNA damage in the whole genome

• transcription coupled repair: NER pathway responsible for recognizing DNA damage in the transcribed strand of active genes

XPG nicks 3’ of damage; XPF nicks 5’ of the damage

repair is completed by pol epsilon or pol-delta RFC, RPA, and PCNA

RPA

replication protein A

TFIIH

complex of proteins including XPB and XPD to unwind DNA double helix and allow for damaged strand to be released

XPF/ERCC

excision repair cross complementing rodent repair deficiency also important for homologous recombination

Global Genome Repair (GG-NER)

NER pathway responsible for recognizing DNA damage in the whole genome accomplished by RPA, XPA, XPC, TFIIH complex

• XPC rapidly scans DNA for structural distortions and opens up DNA after recognizing the target thymine-thymine dimer

  • indirect damage sensor

• pauses when it finds mismatched base to verify a relevant lesion exists before making erroneous incisions

• flips out damaged bases

Transcription Coupled Repair

NER pathway responsible for recognizing DNA damage in the transcribed strand of active genes

bulky lesion halts progress of polymerase and another pair of proteins appear to contribute to the recognition

XPG

an endonuclease

nicks DNA 3’ of the damage

XPF

an endonuclease

nicks the DNA 5’ of the damage

Double Strand Breaks and Repair

breaks are induced by things like reactive oxygen species, ionizing radiation such as X-rays, and chemicals that generate reactive oxygen species

repaired by:

• homologous recombination - high fidelity, requires template, typically restricted to late S-G2

  • major role in prokaryotes and lower eukaryotes

• non-homologous end joining - error prone, template-free, functions throughout the cell cycle

  • somatic cells of higher eukaryotes including mammals

both mechanisms start with detection of the break and subsequent DNA processing

Non-Homologous End Joining

requires heterodimer KU and the catalytic subunit of the DNA-dependent protein kinase, DNA-PKcs

DNA ligase 4-XRCC4 complex ligates the DNA

Homologous Recombination

repairs double strand breaks by getting genetic info from an undamaged homologue (sister chromatic or homologous chromosome) and this requires DNA sequence homology

ataxia telangiectasia mutated (ATM) is the key signal transducer

most insight about this comes from E. coli (prokaryote) and S. cerervisiae (eukaryote) - not the only homology-directed repair, other includes single-strand annealing (SSA)

Steps of Homologous Recombination

1. initiation

2. resection

3. strand invasion

4. DNA synthesis

5. branch migration

6. holiday junction resolution

Initiation of HR

MRN complex = MRE11, RAD50, NBS1 → binds to broken ends of the DNA

recruits ataxia telangiectasia mutated, a serine threonine kinase that phosphorylates proteins involved in the pathway

Resection of HR

starts by MRN complex (MRE11, RAD50, NBS1)

single-stranded 3’ tails are rapidly bound by RPA

Strand Invasion in HR

nucleoprotein filaments subsequently form after replacement of RPA by RAD51 

• nucleoprotein filaments promote invasion of the undamaged homolog, strand displacement, and D-loop formation

displacement of RPA is facilitated by BRCA1-BARD1-PALB2 and BRCA2-DSS1 complex

DNA Synthesis in HR

displaced strand pairs with the broken and abandoned strand. making a heteroduplex molecule and a Holiday Junction

synthesis using the undamaged strand as a template occurs, involving PCNA, DNA pol-epsilon, pol-delta

at the end of synthesis, a double Holiday junction is formed

Holiday Junction Resolution in HR

the resolvasome (RuvABC)

non-crossover (default pathway in S. cerevisiae) requires BTR complex (BLM, TOPIIIa, RMI1-RMI2)

The Holiday Junction

distinct intermediate in which the two recombining duplexes are joined covalently by single-strand crossovers

junction is resolved into two duplexes by an enzyme complex called the resolvasome 

• mammals and yeast: holiday junction may be dissolved of this intermediate in 3 ways

  • non crossover only

  • unbiased (leads to crossover and non crossover)

  • cross over specific - only in meiotic cells