Non-Bulky DNA Damage and Repair

• Focus is on Abasic sites, Abnormal bases, and Single strand breaks (SSBs), which are non-bulky DNA damage

Causes of Non-Bulky DNA Damage

• Can be endogenous, like reactive oxygen species, DNA instability, and enzyme errors
• Can be environmental, such as sunlight, hydrocarbons, and ionizing radiation

Cellular Responses to DNA Damage

• DNA repair
• Genome instability and mutation
• Cell cycle checkpoints, including arrest of mitosis to prevent mutation manifestation
• Cell death

DNA Repair Mechanisms

• Base excision repair (BER) or single-strand break repair (SSBR)
• Mismatch repair (MMR)
• Nucleotide excision repair (NER)
• Double-strand break repair (DSBR)

DNA Damage and Disease

• DNA repair issues can cause cancer and neurological diseases
• Benzo-a-pyrene diol oxide, generated by smoking, is modified by the body into a DNA-damaging product that causes bulky damage
• Benzo-a-pyrene diol epoxide is repaired by NER, but can lead to base loss or abasic sites
• Activation of DNA damage response occurs in early stages of carcinogenesis, even before carcinogenic cells are detectable

Frequency and Susceptibility of DNA Damage

• DNA is damaged 10,000-100,000 times per day
• DNA, as a negatively charged molecule, is susceptible to attack by ions
• The pentose ring in DNA is also susceptible to damage, leading to SSBs
• DNA damage can be spontaneous or induced, with random damage occurring due to DNA's nature

Types of DNA Base Damage & Mutation

• Important to know how DNA damage is converted to a mutation

Base Substitution

• DNA damage can be reversed, but mutations cannot
• DNA damage repair reduces the incidence of damage
• Base substitution is mainly spontaneous
• Deamination, especially in 5-Methylcytosine, can occur higher in 5-Methylcytosine
• 5-methylcytosine is an epigenetic marker, with 5% of cytosines methylated

Point Mutations

• Base substitutions can result in point mutations
• Transition mutation: change of one pyrimidine (C or T) to the other pyrimidine (T or C), or one purine (G or A) to the other purine (A or G)
• G:C to A:T and A:T to G:C are transition mutations
• Transversion mutation: change of a pyrimidine (C or T) to a purine (A or G), or vice versa
• Uracil is read as thymine
• C to U is a pre-mutagenic event
• After cell cycle, adenine that was added to U gets T added to it, therefore we go from G:C to A:T
• Cytosine and thymine are pyrimidines

Base Alkylation

• Can be spontaneous and induced by cellular and environmental alkylating agents
• Monofunctional agents link to one nucleophilic center in DNA
• Bifunctional agents link to two nucleophilic centers in DNA, causing cross-links, which are cytotoxic

Cellular vs Environmental Alkylating Agents

• Cellular: S-adenosyl methionine (SAM)
• 6O-methylguanine is primary mutagenic lesion which mispairs with thymine, resulting in G:C to A:T transitions
• Environmental: anti-cancer agents (e.g. temozolomide) and genotoxins (e.g. NNK, benzo(A)pyrene/BPDE, aflatoxins)
• SAM is endogenous alkylating agent used to drive methylation events
• Temozolomide is a monofunctional methylating agent, used against brain cancer
• Bulky guanine can lead to guanine-sugar backbone loss and subsequent base loss
• BenzoApyrene is super carcinogenic

Base Loss (Abasic Sites)

• Spontaneous and induced
• Instability and hydrolysis of N-glycosyl bond

Types

• Depurination: tens of thousands/cell/day
• Depyrimidation: hundreds/cell/day
• Environmental alkylating agents induce this, such as aflatoxin and BPDE

Characteristics of Abasic Sites

• Abasic sites are both toxic and mutagenic
• Polymerase doesn’t know what to do, can do nothing (toxic) or throw in random base (mutagenic)
• Purines lost more than pyrimidines
• DNA will either leave abasic site empty or with a completely random base

Base Oxidation

• Spontaneous and induced
• Oxygen radicals produced by aerobic metabolism
• X-Rays will damage the sugar-phosphate backbone via radicals
• Radon gas is naturally radioactive, so therefore a risk too

Transversion Mutations

• 8-oxoguanine (8-oxoG) is byproduct of base oxidation
• 8HG can pair with adenine, instead of cytosine, leading to GC to TA base pair, transversion mutation
• Most common mechanism of transversion mutations in cells

Common Sources of SSBs

• Attacks to deoxyribose usually
• SSBs can be an unwanted byproduct of very important processes or caused by torqued stress in the dsDNA band ahead of moving RNA poly
• Some enzymes can deal with torqued stress by allowing break and fixing it
• Treatments can target the TOP1 isomerase in cancer
• Okazaki fragments can be repaired incorrectly, and can cause SSBs
• Reactive oxygen species can attack sugar backbone and cause SSBs
• If you remove a sugar, you normally remove the base too, BD and SSBs are therefore similar

Double-Strand Breaks (DSBs)

• Chromosomes can fragment, thus VERY bad for chromosomes (can lead to complete loss of DNA mutations and aneuploidy)
• Efficient DSB repair mechanisms are needed, such as non-homologous end joining (NHEJ) and homologous recombination (HR)

Processes Creating DSBs

• ~10 DSBs per day per cell
• DSB is most dangerous type of DNA damage because most cells cannot survive even one DSB without repair
• If DSBs are left unrepaired or are repaired inaccurately, mutations and/or chromosomal aberrations are induced and may lead to cell death or cancer

Spectral Karyotyping (SKY Analysis)

• Differential fluorescence labeling based on DNA sequence can show defects in DNA repair (BRCA1/2, BLM..) and defects DNA damage checkpoint (p53, ATM, ATR..)
• BRCA1 or 2 are involved in these processes
• Removing BRCA2 causes abnormal karyotypes, with trisomy, polypleiody and abnormal N numbers, fragmented chromosomes, and fusion across chromosomes
• Translocations can create new allele types

How DSBs Arise

• Physiological DSBs are part of normal cellular process
• Non-physiological/pathological DSBs are exogenous or from errors
• With NHEJ, any base loss can be bad and lead to mutations
• NHEJ doesn’t require homology and DNA similarity, so more prone to errors than HR
• HR uses other DNA template for copying information to repair break and produce high fidelity repair process

Examples of DSB Repair Mechanisms

• Nuclease-induced DSBs (HR or NHEJ repair) like yeast mating-type switching
• Budding yeast can be used to study HR repair mechanisms
• Restriction enzyme (HO endonuclease) can be used to generate DSB in yeast

Homing Endonucleases

• Super specific endonuclease like HO or I-SceI
• CRISPR guide RNAs will target CAS9 endonuclease to specific site in viral genome and generate DSB
• Can be used to modify genome and correct mutations, but raises ethical issues

V(D)J Recombination in Immunoglobulin Genes

• DSBs created within immunoglobulin genes by RAG1/RAG2, this is essential process that creates antibody diversity during immune development
• RAG1/2 create transient hairpin-capped DSBs that are processed using Artemis (opens the hairpin), then fragments are ligated using NHEJ pathway

Meiotic Recombination

• DSBs created ONLY during meiotic prophase by Spo11 enzyme (related to an ancestral form of topoisomerase enzymes (Top6A))
• Spo11 creates a transient covalent protein-linked DSB and must be removed prior to DNA repair
• Generates genetic diversity in gametes and facilitates reductional chromosome segregation during the meiosis I nuclear division
• Spo11 makes DSBs on cells undergoing meiosis and becomes transiently attached to ends of DSBs

Topoisomerases

• Allows for long polymers to exist
• Important when polymers become corded or knotted
• To unknot two coils, Topoisomerases make transient breaks and enable decatenation

Type-II Topoisomerases

• DNA binding: Topo II binds the G-segment (“gate” strand)
• Capture of T-strand: The clamp (yellow) captures another strand (the T “transfer” strand) that will pass through a break made in the G strand.
• Introduction of a DSB: In the presence of Mg2+, the enzyme cleaves the DNA, forming a phosphotyrosine linkage between the 5’ DSB ends of the G-strand and a catalytic tyrosine present in each Topo II subunit.
• Strand passage: The T-strand passes through the break made in the G “gate” strand.
• Resealing the DSB: The transferred (“T”) strand exits the enzyme through the carboxy terminus. The broken “G” strand is resealed.

Characteristics of Topoisomerases

• Very important in Mitotic division
• Generates transient DSBs during DNA metabolic processes: DNA replication, transcription, chromosome segregation, DNA decatenation (unknotting)
• Homologous recombination (HR) is essential for life in higher eukaryotes
• DNA replication errors can be seen after DNA replication
• Chromatid breaks (DSBs) arising during DNA replication require Rad51 for HR repair

How DSBs Arise During DNA Replication

• When the DNA replication fork encounters nicks, covalently attached DNA topoisomerase I, nucleotide aberrations/lesions (UV photodimers), and complex DNA “structures” such as palindromes
• A nick is any type of SS break, base damage or anything
• Nick can then cause polymerase producing laggings strand where laggings strand falls off
• Repaired via HR and Rad51

How Topoisomerases Prevent DSBs

• Relieve topological problems/tension within the DNA double helix, such as supercoiled DNA ahead of the replication fork/transcription bubble, and catenation (linking)
• Create transient DNA breaks, either a single-strand (TopoI) or double-strand (TopoII) of DNA
• Re-ligate the cut DNA ends back together Non-canonical topoisomerase issues can lead to persistent DSBs, can be exploited in cancer therapies

TDP2 and Repair of Failed Topo-II

• TDP2 is enzyme that recognizes stable links, removes phospho-tyr bond and makes clean DNA ends that can be used by the cell
• If cell expresses TP2, it can be very sensitive or not to this

DSB Assays

• Pulsed Field Gel Electrophoresis (PFGE) allows the separation of large chromosome-sized DNA fragments (radiation induces DSBs in DNA, enabling large molecules to enter the gel)
• Cellular survival assays (indirect)
• g-H2AX foci analysis; the rate of foci disappearance correlates with DSB repair
• Whole-genome mapping of DSBs

g-H2AX Foci Analysis

• Measure of the rate of DSB repair with close 1:1 relationship between DSB induction and g-H2Ax foci formation
• DSBs trigger the kinases ATM and ATR to phosphorylate the histone variant, H2AX
• Can monitor kinetics of DSB repair after low IR doses
• ATM phos H2AX, in WT cell, phos histone is removed

Whole-Genome Mapping of DSBs

• Use the power of next-generation sequencing to map the location of DSBs (and SSBs)
• Method: END-Seq (DSB-end sequencing)
• TOP2 activity associated with sites of TOP2B binding and is colocated at regions of strong CTCF binding, a “genome-organising” factor
• Method: CC-Seq (covalent-complex sequencing); TOP2 activity is associated with transcriptionally active locations (marked by histone H3K4 tri-methylation and H3K27 acetylation)

Nucleotide Excision Repair (NER)

• Need to observe the purified protein of NER in context, and relate a mechanism of a defect back to a disease and show how it works

Human Proteins and Orthologues

• Human proteins have orthologues from other organisms (used to identify common processes between species such as NER)
• RAD3 from yeast and XPD in humans

Beginning, Middle, and End of NER

• Beginning: detection of bulky lesion in genome
• Next: recruitment of machinery that fixes damage.
• Final: repair the damage
• Bulky lesion caused by UV, and other dangerous chemicals, can cause cross links
• A bulky lesion can be picked up by XPC-HR23B or XPE-DDB1

DDB Dimer

• Heterodimer that binds to DNA
• DDB is part of large ubiquitin ligase complex; It binds and then ubiquitinates chromatin proteins, recruiting XPC, also self-ubiquitinates, which leads to its degradation
• DDB is good at CPDs but not good at 6-4 photobreaks
• XPE (DDB2) is absolutely required for GG-NER of CPD

XPC Dimer

• Binds flipped out bases on the undamaged strand
• More general than DDB complex

XPD, XPB and TFIIH

• XPD highly homologous to Rad3, well studied, ATP-dependent DNA helicase
• TFIIH has dual functions, in NER and in transcription, so these products are subunits of TFIIH
• XPD and XPB: Helicases that are opening DNA

XP and TTD

• These genes associated with TTD encode subunits of TFIIH
• TTD proposed to be a “transcription syndrome”
• Sites of XPD mutations differ between two syndromes

Structure of Archaeal XPD

• 3 types of mutations that arise from the same gene XPD gives diseases
• Mutations in XP are in catalytic domain
• TTD mutations destroy stability of protein and lead to unfolding
• CS mutation is a hybrid between the two, has helicase activity deficiency, and slight unfolding of the protein too

XPA

• Zinc finger protein, binds more strongly to UV-irradiated DNA, homologous to yeast Rad14
• Binding to UV-irradiated DNA increased by RPA
• XPA and XPC knockouts - mice sensitive to UV light and susceptible to UV carcinogenesis

XPG, XPF-ERCC1

• XPG: structure specific nuclease, suitable for cutting 3' to damage
• ERCC1 and XPF protein heterodimer is a nuclease, cuts 5’ to damage
• XPA double check to see if something IS a lesion, as you don’t wanna waste time
• Rpa coats bubble and prevents rebinding of dna strands to each other
• XPG on 3’, XPF on 5’ side
• Replication proteins (DNA pol, ligases etc) occur after cutting to repair

Transcription Coupled Repair (tcNER)

• Different to general global NER (ggNER) because of the recognition steps
• Have to be actively turning over lesions in genes as, if we don’t, transcription will produce errors
• RNA pol stops at bulky lesion, recruits TRCF, which extradites RNApol from site and recruits AAB complex and repairs lesion
• tcNER identifies lesions from TRANSCRIPTION, as RNA pol stops, whereas ggNER recognizes based on recognition proteins XPD and XPB
• Two factors CSA and CSB recognize lesions in tcNER, and are super important in recognizing lesions in transcription

NER Summary

• XP proteins are involved in damage recognition, unwinding of the DNA and cutting on either side of the damage
• XPD, XPB and TTDA are subunits of TFIIH, which has dual roles in NER and transcription (TTD is a transcription syndrome)
• CS proteins are involved in recruiting NER proteins and chromatin remodelers to enable NER to take place at sites where RNA polymerase is stalled at damage

DSB Repair and Challenges

• Most damage affects one strand, while DSBs affect both strands (no local repair template) (DSBs break the DNA sequence and chromosome-continuity)

DSB Repair Requirements

• Fix the genetic information (restore the nucleotide sequence around the DSB)
• Restore chromosome structure and function to avoid gross chromosomal instability (DSB repair must restore not only continuity but also the sequence itself)

DSB Repair Pathways

• Homologous Recombination (HR): uses complementary strands to exploit sequence homology and copy back info back to the repair site
• Non-Homologous End Joining (NHEJ): ligates together the break ends

NHEJ Discovery

• Cells rely on minimal sequence similarity so that the sequences can pair where they match and a 2 nucleotide deletion occurs (repairs DNA without ensuring the two strands are meant to be together)
• Showed that mammalian cells have the ability to coordinate DNA-break ends which have no significant sequence homology (these non-homologous ends are simply ligated, hence the term Non-Homologous End Joining.)
• Incompatible sequences at new DNA joint can be removed before ligation, resulting in fixed (permanent) mutations (this is referred to as error-prone repair) Error-prone NHEJ is useful for CRISPR/Cas9-mediated gene disruption

HR and NHEJ vs Cell Survival

• Both HR and NHEJ-mediated DSB repair protect cell viability
• Deficiency of BOTH types of DSBr is very bad, but just one is not lethal, just bad.

NER Summary

• Proper DSB repair in cells requires HR and NHEJ (NHEJ provides quick DSB repair at cost of repair fidelity)
• NHEJ appears to be a trade-off between speedy DSB repair and repair fidelity, promoting cell viability in the face of toxic DSBs, but increased risk of mutation
• Context matters. Cells can steer endogenous DSBs towards homologous recombination repair

Key Proteins of NHEJ

• PNKP will cause changes between 3’PO4 and 5’OH and convert one to another. Either way. Can do 3’PO4 to 5’OH or 5’OH to 3’PO4
• If you have a 3’ PO4 with bulky adduct, you’d need TDP1 to remove the adduct and prep the 3’ PO4
• A 5’ PO4 with an adenosine residue attached will be processed by aprataxin to produce a smooth 5’PO4 end
• Pol gamma and mu will fill gaps and enable ligation of the sequence
• TdT will insert a nucleotide and leave room for NER and ligation
• Artemis will remove a hairpin loop and enable ligation and other factors to come in (core factor)
• Ku70 and Ku80 sit at the end of the break and perform end-binding, assembling the repairosome (these can then be processed by phos Artemis)

Key Complex of NHEJ

• XECC4-LIG4-XLF is ligation complex (unidirectional)

The Ku70-Ku80 Heterodimer

• Has a preference to bind dsDNA ends and is super abundant with very high affinity for DNA
• Associates with DNA-PKcs, forming a DNA-dependent kinase

DNA-PKcs

• Consists of HEAT and kinase domains
• Stimulated by linear, not circular, DNA
• Only works as an ensemble of DNA-PKcs with Ku70/80
• Form a DNA dependent kinase that can phos other proteins to activate them (switched on when dsDNA breaks present)
• Assembles on, and protects, DNA ends; its kinase activity is regulated by the PRD
• Autophos locks the complex into active state, enabling processing of the ends (end becomes exposed, allowing access of end-processing/ligation factors) and conformational changes determine kinase activity
• Artemis works in the context of the ku70/80 and DNA pk complex
• Artemis is required for NHEJ at a subset of (“untidy”) DSBs (DSB repair curves after IR are biphasic; most breaks easily ligatable, but some are hard to ligate simply, so artemis used to finish these off)
• DNA-PK acts as a scaffold for DSB end-processing (Artemis, X-family polymerases)

X-Family Polymerases

• Mediate the maturation of ends during NHEJ (can perform gap filling step)
• Cells that express homologous –ve pol ƛ show worse outcomes always
• Expression of a catalytically inactive form of Polλ (polλDN) results in increased sensitization and genomic instability in response to ionizing radiation

DNA Ligase IV Complex

• Ligation of DSB ends; no template available
• Synapsis must occur in order to repair ends (requires complex)
• The Ligase complex organises end-synapsis during DSB repair (interactions between XLF and XRCC4 are present, and also other interactions with Ku complex)
• Requires WT proteins throughout, not enough for just ligase to bind, same with Ku and XLF (needs entire assembly to bring this process about)
• A structural role of Lig4 in close-range synapsis may promote FIDELITY (structural interactions of Lig4 may help prioritise ligation of compatible ends over end processing)

NHEJ Summary

• DSBs affect local DNA sequence and structure/function of chromosomes
• Cells use error-free HR and error-prone NHEJ to fix DSBs and protect cell viability
• NEHJ provides quick DSB repair at the expense of repair fidelity
• KU avidly binds DNA ends and associates with DNA-PKcs to form an active DNA-PK at DSBs
• DNA-PK acts as a scaffold for DSB end-processing (Artemis, X-family polymerases)
• XRCC4-Lig4-XLF mediates short-range synapsis, priorities ligation, and promotes NHEJ fidelity