M1L6 DNA replication defects

  • Enzymatic and non-enzymatic DNA-protein crosslinks (DPCs) are a poorly studied form of DNA damage

  • DPCs are a result of covalent attachment of proteins to DNA (high probability due to large number of proteins associated with DNA - histones, replicative enzymes, transcription factors, DDR proteins etc…)

  • Guanine and lysine are the strongest cross linkers

    • There are many lysine residues in histone tails, making them prone to attach to guanines in DNA

  • Formaldehyde is a major inducer of DPCs, as well as chemotherapy drugs (eg. topoisomerase inhibitors, cisplatin…)

    • Topoisomerase binds to DNA to make transient SSBs in DNA to relieve torsional strain, inhibitors stabilise this complex leading to propagation of DSBs

  • DPCs: non-enzymatic and enzymatic

    • Non-enzymatic: formaldehyde and methanol

      • Aldehydes (formaldehyde, acetaldehyde) can form covalent bonds between amino acid residues and DNA bases

      • UV or IR can also cross link nearby proteins to DNA

      • Endogenous metabolism can product reactive aldehydes and lipid peroxidation byproducts that contribute to this

    • Enzymatic: lesions caused by topoisomerase, DNA polymerase β etc enzymes

    • Stalls replication fork

  • Ubiquitination and SUMOylation mark DPCs for degradation

    • DPCs can be resolved through proteolysis or HR if fork collapse leads to DSBs or if there are DSBs resulting from topoisomerase activity

    • Proteolysis is carried out by the proteasome and SPRTN protease which degrade protein adducts

    • After partial proteolysis repair is completed by TLS polymerases which bypass the remaining peptide adducts and NER to remove residual protein fragments

  • Formaldehyde can cause many types of DNA lesions, mostly triggering covalent attachment of lysines of proteins to guanine in DNA (DPCs)

     

  • Ub and its chains coordinate spatial and temporal regulation of proteins at DNA damage sites

    • Eg. Ub at DNA DSB sites

    • MRN detects the DSB and recruits/activates ATM kinase

    • ATM phosphorylates H2AX near the break

    • MDC1 binds to γH2AX, stabilising the repair focus

    • ATM phosphorylates L2MCTL2 which interacts with MDC1 and RNF8 (an E3 ubiquitin ligase)

    • RNF8 ubiquitinates histones and other local proteins

    • RNF8-generated ubiquitin chains recruits RNF168 which amplifies ubiquitin signalling my spreading ubiquitin marks on chromatin

    • Ubiquitin marks act as docking sites for repair proteins like 53BP1 (for NHEJ) or BRCA1 (for HR)

    • For NHEJ:

      • 53BP1 binds to ubiquitylated H2A/H4

      • HR is blocked as 53BP1 and KEAP1 inhibits BRCA1 recruitment

      • PTIP FIF1, shieldin complex, CST–Polα–Primase, and Artemis assemble, processing and ligating DNA ends directly

    • For HR:

      • BRCA1 is recruited by RAP80, RNF8/RNF168

      • BRCA1 antagonises 53BP1 and promotes end resection for HR

      • MRN, CtIP, EXO1 and DNA2 create 3’ ssDNA overhangs 

      • RPA coats ssDNA and BRCA1/PALB2/BRCA1 load RAD51. forming the nucleoprotein filament

      • RAD51 filament searches for homologous DNA template and promotes strand invasion and HR repair

    • Human genetic disorders associated with inherited mutations in various E3 ubiquitin ligases involved in DNA replication/repair - usually cancers, neurodegeneration, age-related pathologies but also others

    • Ubiquitination must also be removed for resolution of DSBs, eg. inhibition of p97 (which helps remove ubiquitination) does not allow for full resolution of DSBs 

  • Chromatin associated degradation (CHROMAD)

    • p97 (aka VCP) is an ATPase in the ubiquitin system which recognises ubiquitinated substrates and uses cofactors with specificity to the substrate to bind and hydrolyse ATP and thereby extract substrates from chromatin

    • p97 is a homohexamer and physically extracts substrates through its central pore

    • Cofactors of p97 are implicated in human disease -

      • Ataxin-3 (mutated in ataxia/Machado-Joseph disease) which works with p97 to remove proteins by the proteasome, specifically RNF8 

      • SPRTN (mutated in Ruijs-Aalfs Syndrome - premature ageing) - important for removing DPCs

      • TEX264 (colorectal cancer)

  • Formaldehyde (from the environment or due to our own metabolism) is a cause of ageing pathologies including neurodegeneration and cancer

    • Formaldehyde can be from smoke, industrialisation, environmental stressors but also a product of endogenous metabolism:

      • Synthesis of some amino acids or nucleic acids

      • Methyl groups removed by demethylases can produce formaldehyde in the presence of oxygen

      • Mitochondrial stress causes lipid peroxidation in its double lipid membrane, producing formaldehyde

    • Endogenous formaldehyde causes new inherited bone marrow failure syndrome (short stature, microencephaly, limb abnormalities)

  • Two-tiered protection from aldehyde (acetaldehyde and formaldehyde) toxicity

    • Detoxifying enzymes - aldehyde and alcohol dehydrogenases

    • DNA repair (DPCs)

  • Ethanol metabolism

    • Alcohol dehydrogenase converts ethanol to acetaldehyde (toxic) by using NAD+ which gets converted to NADH + H+

    • Acetaldehyde is converted to acetate by aldehyde dehydrogenase using NAD+ which gets converted to NADH + H+

    • Acetate is converted to acetyl CoA by acetyl CoA synthetase 2

  • Methanol metabolism - methanol can come from consuming cheap alcohol (methanol mixed with ethanol) or even from your own microbiome via fermentation processes

    • Methanol converted to formaldehyde by alcohol dehydrogenase (very toxic)

    • Formaldehyde converted to formic acid by formaldehyde dehydrogenase —> metabolic acidosis + tissue injury

      • Inhibiting ADH5 can lead to high levels of formaldehyde toxicity

    • Formic acid converted to CO2 + H2O

  • Formaldehyde induced DPCs

    • Tier 1 defence - ALDH2, ADH5

    • Tier 2 - DPC proteolysis repair, Ub system (SPRTN, p97, nucleophagy)

  • Human diseases caused by endogenous DPCs (likely due to formaldehyde toxicity)

    • AMeD syndrome (aplastic anaemia, intellectual disability, dwarfism) - digenic mutations in ALDH2 (SNP; on one allele) and ADH5 (biallelic mutations)

    • Ruijs-Aalfs syndrome - monogenic cause (biallelic mutations in SPRTN)

    • Cockayne syndrome - defect TC-NER, monogenic and biallelic mutations in CSA or CSB

  • SPRTN is key enzyme (metalloprotease) in DPC repair, needed for progression of replication fork

    • Cleaves a variety of DNA binding substrates (eg histones) and also itself

    • Cleaves free and covalently attached Top1 and Top2

    • Defects in SPRTN characterised by RJALS and liver cancer

  • p97-SPRTN-TEX264 complex processes Top1-cleavage complexes (DPCs)

    • TEX264 is one of the main receptors for autophagy during starvation and it also plays a major role in DPC repair by acting as a p97 adaptor protein

    • p97-SPRTN-TEX264 can recognise DPC lesion (eg. Topo1cc or Topoisomerase 1-cleavage complexes which is an enzymatic DPC) on DNA

    • TEX264 recruits p97/SPRTN to the DPC and SPRTN proteolytically degrades the protein adduct while p97 proides unfolding and extraction energy to clear the protein remnants from chromatin

    • After DPC processing DNA can be repaired by DDR mechanisms

    • TEX264 also acts as an ER-resident autophagy receptor where it interacts with LC3 on phagophores for ER-phagy

  • Nucleotide excision repair

    • UV lesions, formaldehyde induced lesions…

    • Transcription-coupled NER:

      • RNA pol II stalls at a lesion

      • CSB binds to stalled RNA pol II and recruits CSA (E3 ubiquitin ligase) and other repair factors, remodeling RNA pol II and forming the damage recognition complex that distinguishes transcription blocking lesions 

      •  TFIIH recruited to the site which containes the helicases XPB and XPD to unwind local DNA

      • XPG (endonuclease) and other XP proteins (XPA, XPB, DPD) join to form the NER core complex

      • SPG and XPF-ERCC1 make dual incisions flanking the lesion and the damaged oligonucleotide is excised

      • RPA and XPA stabilise the single stranded region\

      • XPA is also an E3 ubiquitin ligase which ubiquitinates TC-NER proteins like CSB and TFIIH to regulate their recruitment, retention or release at the lesion site

      • The gap is filled by DNA pol using the undamaged strand as a template and the nick is sealed by DNA ligase

      • After repair XPA and othe ligases promote proteasomal clearance of excess or damaged repair factors to prevent persistent stalling

    • TC-DPC repair begins as a TC-NER process

      • RNA pol II stalls at DPC and CSB binds and recruits CRLRCSA ubiquitin ligase complex which ubiquitinates the DPC to mark it for degradation and also RNA pol II to mark it to be removed or recycled after the lesion is repaired

      • p97 ATPase unfoldase is recruited to extract/unfold the ubiquitylated DPC protein from DNA

      • TFIIH likely facilitates or stabiilises p97 recruitment and activity but is not essential for unfolding itself, rather helps remodel RNAPII-DNA-DPC complex to improve access for p97

      • DPC protein is then degraded by the proteasome

      • TFIIS (transcription elongation factor) helps restart or rescue RNAPII

      • Remaining peptide adducts could be handled by SPRTN (like in RC-DPC repair)

  • Parallel DPC repair pathways

    • RC-DPC (SPRTN) -replication coupled

    • TC/DPC (CSA/CSB) - transcription coupled

    • Both converge on p97 (VPC)

  • Formaldehyde inhibits DNA replication

  • Top1 cleavage complexes induced by Top inhibitors and PARP1 complex induced by PARPi

  • Selective autophagy (nucleophagy) of Top1cc

    • Top1cc removal is strongly dependent on autophagy (DPC lesion-phagy)

    • May explain why many cancers upregulate autophagy

    • Part of the DPC lesion which can not be removed by SPRTN are becoming insoluble and forming aggregates

    • In this case, TEX264 removes the lesion and shuttles it to the lysosome for degradation

    • When there are DPCs autophagy proteins and lysosomes can be recruited to the replication fork even from outside the nucleus

      • There is interorganelle communication - replication fork recruits lysosomes and lysosomes can also recruit replication proteins

  • TEX264 expression can be an predictor of responsiveness to Topo inhibitors (higher expression = better responders)

  • Nucleophagy can also remove cytotoxic trapped PARP1

  • Inactivation of TEX264-selective autophagy resensitises BRCA-deficient cells that have acquired resistance to PARPi