M1L2 DNA damage and repair

What causes DNA SSBs?

Radiation and oxidative stress

What causes DNA mismatches?

Replication errors, homologous recombination, cytosine deamination to uracil, 5meC deamination to thymine, adenine deamination to hypoxyanthine (codes like G)

What causes damage to nitrogenous bases?

radiation, oxidative species, alkylation (particularly methylation), deamination

What causes abasic sites?

Occurs spontaneously, particularly for purines, especially if the base is alkylated, also an intermediate in the action of DNA glycosylases

What gives rise to altered based?

Pyrimidine dimers, alkylated DNA, oxidised based

What causes DNA DSBs?

Ionising radiation

What produces intra-strand crosslinks?

UV light and cancer drugs

What produces inter-strand crosslinks?

Cancer drugs and metabolites (eg aldehydes)

What are the main types of DNA mutagenesis?

Deletions, insertions, substitutions

Transitions - pyrimidine substituted for a different pyrimidine or purine substituted for another purine

Transversion - pyrimidine substituted for purine or purine substituted for pyrimidine

What is the role of photolyase in direct repair in bacteria, fungi, plants, and certain animals?

• Strong affinity for structure of damaged lesions and can reverse base damage caused by UVB radiation

  • Pathway has two cryptochromes (FAD and FADH^-) which can absorb blue light (300-500nm)

  • FADH^- gets excited, transferring an electron to the UV damaged site

  • Breaks chemical bonds causing DNA damage and returns pyrimidines to original configuration

How are O⁶ alkyl groups directly repaired by the suicide protein O⁶-ATase?

• O⁶ alkyl guanine is recognised by the ATase which transfers the alkyl group to a cysteine residue in its active site, causing covalent attachment of alkyl group to ATase

• This corrects the damage but inactivates the ATase, hence protein turnover requires proteolysis and resynthesis

How is O⁶-ATas implicated in drug resistance?

• Temozolomide induces DNA methylation in the treatment of brain cancers, however cancer cells tend to upregulate this ATase as a resistance mechanism

  • Drug development investigating inhibitors of these ATases to prevent this, but there are issues with bypassing the blood brain barrier and tumour evolution

What are two pathways that can be used to repair O⁶-alkyl guanine?

Direct repair and base excision repair

What are the three broad steps of BER?

• Lesion recognition

• Removal of damage nitrogenous base

• Short/long patch BER

How are DNA lesions recognised in BER?

• The enzymes have a motor protein that slides rapidly up and down the double helix

• Damaged base can be recognised by shape, hydrogen bonding potential and electric charge distribution

• When sliding along the phosphate backbone they can flip out bases from the interior of the helix

• Once flipped into the enzyme binding pocket, damaged bases but not normal bases get cleaved by glycosylase

• Normal bases will have a short residency time in the active site but damaged bases have a long residency which permits the enzyme to attack the N-glycosidic bond to cleave the base

• Damage can be recognised from seconds to minutes

How are damaged bases removed in BER?

• Initiated by monofunctional glycosylases which have high specificity for small chemical base modifications (eg deaminated cytosine, oxidised guanine…)

• Nitrogenous base gets removed by glycosylase, producing an AP site (abasic site)

• AP endonuclease cleaves the AP site, exposing a 3’-OH on one side of the cleavage site and deoxyribose phosphate (dRP) on the other side

Describe short patch BER

• DNA pol β (has dRP lyase activity) removes the phosphorylated ribose to restore normal DNA biochemistry by exposing 5’-phosphate but leaves a 1-nt gap

• Pol β extension fills the gap and DNA ligase III/XRCC1 seals the nick

Describe short patch BER

• DNA pol β/δ/ε extends from the 3’-OH for 3-12 nt which generates a displaced flap

• Flap endonuclease cleaves the flap and the dRP is thus removed

• Nick is sealed by DNA ligase I/PCNA

What is Xeroderma pigmentosum?

a condition associated with defects in NER causing severe photosensitivity, skin and non-skin cancers, and neurological abnormalities

How is DNA damage recognised in NER?

• Detects bulky, large chemical damage (eg. crosslinked adjacent pyrimidines due to UVB)

• Damage is detected by structural distortion and defamation of the DNA structure as bulky chemical damage alters base pairing and DNA geometry, causing local unwinding/denaturation and lack of base pairing at the damaged site

• XPC recognises the lack of base pairing

• TFIIH is recruited by direct interaction with XPC

• XPB (moving 3’ to 5’) and XPD (5’ to 3’) helicases unwind DNA around the lesion

• XPD particularly acts as a damage verification helicase and moves towards the damage and its unwinding activity is arrested by DNA damage, generating a static bubble which can be cut by the endonucleases

• If there is no damage XPD would continue to translocate along the DNA and continue unwinding which collapses the NER apparatus, avoiding incision

What are the steps of NER?

• DNA damage is recognised by XPC-HR23B and the DNA duplex is opened up

• TFIIH (related to another form of TFIIH involved in initial RNA pol II transcription), XPA (structural role), XPB and XPD helicases (create a bubble structure of DNA) and RPA (single stand binding protein) are recruited to the damaged strand

• Bubble structure creates landmarks whereby endonucleases ERCC1-XPF (cuts 5’ to the damage) and XPG (cuts 3’ to the damage) can excise the strand

  • ERCC1-XPF cuts first, followed by DNA synthesis, and then XPG cuts the flap (rather than a flap endonuclease)

    

• 20-30nt stretch of DNA strand containing the damage is removed, leaving a gap which gets filled by DNA pol ε/δ/κ and PCNA (which is supported by RFC acting as a clamp loader) using the undamaged strand as a template

• The nick is sealed by DNA ligase I/III

How can NER be transcription coupled?

• A subpathway of NER operates independently of XPC-HR23B as it is triggered by RNA pol II arrest at a lesion

• Cockayne syndrome A and Cockayne syndrome B proteins (CS factors) are needed to couple the arrest of RNA pol II to the NER factory

  • Cockayne syndrome A protein is a motor protein which hydrolyses ATP and can move things around on DNA, could possibly push RNA pol II back away from the lesion to allow space for the NER apparatus to work

  • They may be involved in recruited and positioning other NER proteins

• NER on UV damage induced regions are is more efficient in rapidly transcribed reghions of the genome due to RNA pol II

• Cells degrade RNA pol II as a last resort if transcription coupled repair fails which may enable global genome repair function

What happens in Cockayne syndrome?

• Cockayne syndrome - characterised by growth failure, impaired develoopment, photosensitivity, premature aging, hearing loss/eye abnormalities, sometimes associated with Xeroderma pigmentosum

  • Due to defective transcription coupled repair (80% due to defective CSA, some due to defect in core NER factors)

  • Tend to be protected from skin tumours and reach maturity before phenotypic consequences

  • One theory is that these individuals do not deal with RNA pol II stalling well and its breach of DNA damage causes permanent stalling and a gradual breakdown of the gene expression profile

Explain how translesion synthesis (TLS) polymerases enable DNA damage tolerance

• Replicative pol stalls and switches with TLS pol which can accomodate damage due to low processivity and continue extension, leading to mutation, they later switch back with the replicative pol after passing over the damaged region

• Switch from replicative to TLS pol controlled by PCNA modification in eukaryotes

  

  • Upon arrest, replicative pol is removed/remodelled at PCNA, causing monoubiquitination of K164 on PCNA by Rad18 and Rad6

  • Causes recruitment of TLS pols (Pol ι and η) which continues extension and later switches back with replicative pol

  • Deubiquitination of PCNA is time consuming if it at all occurs, so not strictly necessary for re-exchange of TLS pol for replicative pol

  • Pol η has evolved to insert AA opposite TT adduct so does not necessarily cause mutation

What may be the result of TLS defects?

When TLS by Pol η goes wrong it causes Xeroderma pigmentosum variant form, characterised by defect in conversion of newly synthesised DNA from low to high MW after UV irradiation

Describe what is Fanconi anaemia

• ecessive chromosomal instability syndrome

• Bone marrow failure and childhood AML

• Cancer predisposition up to x10,000

• Developmental abnormalities

• Genetically heterogeneous - 22 genes identified

  • FANCA and FANCC mutations (most common) - less severe phenotypes

• Carrier freq ~1:200

• Exclusive hypersensitivity to replication blocking DNA damage exemplified by DNA interstrand cross linking (ICL-inducing) agents

What causes Fanconi anaemia?

• Defects in ICL repair leads to Fanconi anaemia

• Sources - endogenous aldehydes, oxidised lipids… etc

What are the clinical implications of mutations in FANCD1 (BRCA2) or Rad51C?

High penetrance breast cancer susceptibility alleles

What are the clinical implications of mutations in FANCJ or FANCN?

Low penetrance breast cancer susceptibility alleles

What are the clinical implications of mutations in FANCQ (XPF)?

Multiple inherited cancer prone/developmental disorders

What happens in the FA pathway?

• FANKL monoubiquitinates FA ID complex (FANCD2/FANCI)

• FANCD2/FANCI bind to dsDNA around the damaged replication fork and bind to it to recruit other proteins

  • ICL incision, TL synthesis, HR

• But only FANCM (translocase), FANCL (E3 ubiqutin ligase) and XPF (nuclease)

What are the steps of the FA pathway?

• Convergence of replication forks signals the presence of the lesion and triggers FA pathway

  • FANCM may function to rewind the DNA behind the stalled fork (fork reversal) which generates a region of dsDNA adjacent to the crosslink

  

• Disassembly of the replisome (CMG unloading)

• Activation of FA core complex

• FANCI-FANCD2 monoubiquitylation by FANKL

• Recruits SLX4 and ERCC1-XPF to cleave DNA crosslink, releasing one of the damaged chromatids

  • dsDNA adjacent to crosslink produced by FANCM is an excellent substrate for SLX4 and ERCC1-XPF mediated cleavage

  • SNM1A recruited to ERCC1-XPF incised intermediate by interacting with PCNA using its PIP motif

    • This is a damage tolerant 5’-to-3’ exonuclease that digests through and past the incised intermediate, leaving a single nucleotide and providing a downstream template for subsequent TLS complete repair of crosslink chromatid

      
    

• TLS polymerases take the stalled fork and extend it past the cross link in an error prone manner

  

  • TLS pol ζ recruited to ERCC-1 incised and SNM1A-resented ICL and synthesises DNA past ICL, repairing first chromatid

• This produces an intact chromatid and a broken chromatid (DNA DSB) which is repaired by BRCA2 and other HR factors using the intact chromatid as a template

• Remnant of the initial crosslink may be removed by NER