CMMB 411: Topic 6 - DNA Damage, Mutation, and Repair Mechanisms

sources of DNA damage and mutations

1. errors during DNA replication - nucleotides are misincorporated - results in permanent change in DNA sequence after replication - or, can be detected and corrected (but sometimes, repeated errors are difficult to fix) 2. physical damage to DNA - DNA gets chemically altered (bases) - leads to mutation if not repaired - many types 3. mutations (are a balance or a combo of damage + error and repair systems) - permanent changes in the DNA sequence - can be a single base change (point mutation) - or a large change such as an insertion, deletion, or rearrangement

molecular mechanisms that lead to mutations

1. errors in DNA replication 2. base loss 3. chemical modifications 4. action of intercalating agents 5. base analogs 6. double strand breaks

the GO (8-oxo-guanine) repair system in bacteria

2 glycosylases MutM and MutY function in the GO repair system - MutM: a glycosylase; removes GO from A:GO and C:GO base pairs - MutY: a glycosylase; removes mis-incorporated A from A:GO Prediction of mutations resulted from mutM or mutY mutations? mutM- --> G:C to T:A; transversion mutY- --> C:G to A:T transversions

Q4. How does DNA Polymerase slippage contribute to genetic disorders like Fragile X syndrome?

DNA pol slippage happens during replication when the polymerase temporarily dissociates from the DNA template and then reanneals in the wrong position. It often happens in regions w/ repetitive sequences, like the CGG trinucleotide repeats found in the FMR1 gene associate w/ Fragile X syndrome. If slippage happens in these repeats, it could lead to an expansion of the repeat sequence. Over successive generations, the expansions could increase in size, and when they cross a certain threshold, they can result in the gene's inactivation. In the case of fragile X syndrome, too many CGG repeats lead to methylation and FMR1 gene silencing, disrupting production of the fragile X mental retardation protein (FMRP), which is essential for normal neural development, thereby causing the disorder.

Q6. Contrast the repair processes MMR, BER, NER, TLS, and NHEJ vs. DSBR by HR (part 3) - Contrast NHEJ and DSBR by HR

Non-Homologous End Joining (NHEJ): NHEJ repairs double-strand breaks by directly ligating broken ends together, often w/o a template, which can result in a loss or gain of nucleotides at the repair site. Double-Strand Break Repair (DSBR) by Homologous Recombination (HR): DSBR by HR is an error-free repair mechanism that requires a homologous sequence to guide repair, typically using a sister chromatid as a template. It involves strand invasion, template-directed synthesis, and resolution, resulting in accurate repair. Contrast NHEJ and DSBR by HR: NHEJ quickly rejoins DNA ends but can be error-prone, while HR is slower but uses a template for accurate repair, typically w/o introducing mutations. NHEJ operates throughout cell cycle, while HR is mostly limited to S and G2 phases when sister chromatid is available.

chemical modifications (part 1)

a) deamination: removal of an amine group - cytosine to uracil - adenine to hypoxanthine - guanine to xanthine - 5'-Me-cytosine to thymine *** these are difficult to repair b/c natural nucleotide is hard for the cell to distinguish b) oxidation: oxidative metabolism and ionizing radiation produce reactive oxygen species (ROS), such as singlet oxygen, superoxide free radicals and H2O2. execute chemical modification of bases - e.g. ROS can oxidize thymine to thymine glycol (nucleotide which blocks DNA pol); guanine to 8-oxo-guanine c) alkylation: addition of an alkyl group (e.g., CH3) to bases at nucleophilic positions

par 2. repair chemical damage to DNA

examples of DNA damage are as follows: - deamination of C to a U - depurination: hydrolysis of a base - deamination of 5-mC to a T - thymine dimers caused by UV light - sites of possible damage for G - double stranded breaks or DSBs

1. errors in DNA replication

with 3' to 5' proofreading of DNA polymerase, error rate is about 1 in 10^7 bases. it would result in 6000 errors per diploid genome (6.4 x 10^10) per cell division - proofreading improves fidelity of DNA replication by a factor of ~100.

mutation signatures in cancer

C>A enriched in signature - defective base excision repair, including DNA damage due to reactive oxygen species, due to biallelic germline or somatic MUTYH mutations (oxidative damage of guanine)

chemical modifications (part 2)

d) UV-induced damage: by forming cyclobutane pyrimidine dimers (CPD), which distort DNA double helical structure - most common are thymine dimers (T-T); UV light can induce the formation of a cyclobutene ring between adjacent thymidines - dimers can also be produced by formation of single covalent bond b/w 6 position of one pyrimidine and 4 position of the adjacent pyrimidine on the 3' side, known as pyrimidine (6-4) pyrimidine photoproducts (6-4PPs), e.g. T-C; C-C - likelihood of dimer formation: T-T >> T-C > C-T > C-C

spectrum of variants

in human genetics, genetic variants now widely used instead of "mutations" e.g. point mutation --> single nucleotide variant (SNV); tandem duplication; translocation; insertion; interspersed duplication; deletion; inversion; repeat expansion

how do intercalating agents like ethidium cause short insertions and deletions?

one theory is as follows: by slipping between the bases in the template strand of the DNA double helix, these mutagens cause DNA pol to insert an extra nucleotide opposite the intercalated molecule. typically, the intercalation of one of these structures approximately doubles typical distance b/w two base pairs.

direct reversal of damage

photoreactivation: DNA photolyase uses energy of visible light to reverse the pyrimidine dimers that result from UV irradiation. - DNA photolyase enzyme uses visible light energy to break covalent bonds linking adjacent pyrimidines in a fairly quick process methyltransferase removes methyl from a G: - another example of direct reversal is the removal of methyl group from the methylated base O^6-methylguanine - methyltransferase removes methyl group from guanine residue by transferring it to one of its own cysteine residues. *** this is costly to the cell b/c methyltransferase is not catalytic; having once accepted a methyl group, it cannot be used again

Q6. Contrast the repair processes MMR, BER, NER, TLS, and NHEJ vs. DSBR by HR (part 2) - Contrast MMR, BER, and NER. - TLS

Contrast MMR, BER, and NER: MMR targets mismatched bases without distortion, BER corrects small base lesions w/o large helix distorions, and NER repairs large, helix-distorting adducts. Translesion DNA Synthesis (TLS): TLS is a damage tolerance mechanism allowing replication to continue at a stalled replication fork by using specialized DNA polymerases that can synthesize across lesions, albeit with a higher risk of introducing mutations.

1b. "Slippage" of DNA polymerase

- slippage during replication at DNA repeats (di, tri, or tetra nucleotide sequences, e.g. CA, CGG and CAG repeats - large stretches known as DNA microsatellites) - slippage increases or reduces the number of copies of repeated sequences - several diseases are caused by slippage of DNA polymerase trinucleotide repeat disorders: involve expansion of repeats of CAG, CGG, GAA, and CTG, implicated in >30 human hereditary disorders (recorded) such as Fragile X syndrome, Huntington's disease

Q2. Types of SNV (Transitions and transversions)

Single nucleotide variants (SNVs) are changes in the DNA sequence involving substitution of a single nucleotide. There's two main types of SNVs: transitions: substitutions where a purine is replaced w/ another purine (adenine (A) ↔ guanine (G)) or a pyrimidine is replaced with another pyrimidine (cytosine (C) ↔ thymine (T)). transitions are the most common type of SNV. transversions: involve the substitution of a purine for a pyrimidine or vice versa (adenine (A) or guanine (G) ↔ cytosine (C) or thymine (T)). there are twice as many possible transversions as transitions, but they happen less often. both transitions and transversions can have a range of effects on an organism, from benign to causing serious diseases, depending on where they occur and whether they alter the function of essential genes.

5. base analogs

base analogs are compounds that substitute for normal bases to cause replication errors - similar enough to be treated as normal bases and converted to nucleoside triphosphates and incorporated into DNA during replication - base pairs inaccurately due to structural differences between the analogs and the proper bases, the analogs base pair inaccurately, leading to frequent mistakes during replication process

2. base loss

base getting cleared from sugar = base loss - depurination (loss of purine bases) and depyrimidination (loss of pyrimidine bases) - glycosidic bond cleavage (hydrolysis) b/w base and deoxyribose to yield abasic (AP) site. glycosidic bond not 100% stable in aqueous solution. - under physiological conditions, deprivation occurs at ~5000 bases/cell/day - when DNA tries to use strand w/ base loss (ds breaks), the replication is stalled

part 1. fixing errors during DNA replication

the repair system needs to act fast (before next round of replication) and correctly (misincorporated nucleotide on newly-synthesized strand vs. correct nucleotide on parental strand) in the figure, one can see how replication can change a misincorporated base into a permanent mutation - 1st round of replication: a potential mutation may be introduced by misincorporation of a base. - 2nd round of replication: misincorporated base becomes permanent in the DNA sequence and is now a mutation

classes of repair mechanisms

1. to 3. will be the most focussed on. 1. direct reversal - corrects specific damage (e.g. photo activation: pyrimidine dimers via photolyase) 2. base excision repair (BER) - damaged bases; initiated by DNA glycosylases 3. nucleotide excision repair (NER) - repairs bulky adducts on bases and pyrimidine dimers 4. double-strand breaks repair - homologous recombination (HR): error-free, uses sister chromatid - non-homologous end joining (NHEJ): directly joins ends, can be error-prone 5. translesion DNA synthesis (TLS) - specialized polymerases replicate past lesions; can be error-prone. repairs apurinic site, bulky adduct on bases and pyrimidine dimers.

what pathway removes uracil present in DNA? A) base excision repair B) nucleotide excision repair C) non-homologous end joining D) translation synthesis

A) base excision repair base excision repair (BER) is the process by which cells repair damaged DNA during cell cycle. when uracil is present in DNA, typically due to deamination of cytosine, it is recognized and removed by enzyme uracil DNA glycosylase. BER then proceeds to restore correct base and maintain DNA integrity.

human glycosylases

UNG (uracil DNA N-glycosylase): - targets: uracil in DNA from cytosine deamination or misincorporation - substrates: ssU, U:G, U:A TDG (thymine DNA glycosylase): - targets: thymine paired w/ guanine (due to 5-methylcytosine deamination) - main substrate: T:G MPG (methyl purine DNA glycosylase): - targets: alkylated bases in DNA - note: recognizes variety of alkyl groups, not just methyl OGG1 (8-oxo-guanine glycosylase): MutM - targets: oxidative DNA lesion 8-oxo-guanine - substrate: 8-oxo-G - results from oxidative damage MUTYH (MutY homolog): - targets: adenines misfired with 8-oxo-guanine - substrates: A:G, A:8-oxoG NTH1 (endonuclease three homolog): - targets: oxidized pyrimidines - main substrates: thymine glycol, cytosine glycol BER is lesion-specific; different lesions need to be recognized w/ specific glycosylases

6. double strand breaks (DSBs)

- DSBs result from DNA backbones of two complementary DNA strands being broken simultaneously. - lethal to cells if not repaired. exogenous sources: 1. ionizing radiation: X-ray, radon, cosmic rays (strands either broken directly or indirectly by ROS generation) 2. anticancer chemotherapeutic drugs: topoisomerase inhibitors (campthothecins and etoposides can trap topoisomerases during breaks so that they can't reseal properly) endogenous sources: 1. stalled replication forks 2. programmed DSBs: meiotic recombination; mating type switching in yeast

nucleotide excision repair (NER)

- an enzyme scans DNA for distortions from the standard B-form helix (e.g. a thymidine dimer; a bulky chemical abduct on a base) - when damage is found, short patch of nucleotides is removed and the gap is filled - in E. coli, four enzymes are used: UvrA, UvrB, UvrC, UvrD - the dimer of UvrA and the dimer of UvrB form a tetramer that scans a DNA helix - when a lesion is encountered, UvrA leaves and UvrB melts two DNA strands locally around the distortion - UvrC will form a complex with UvrB and cleaves on both sides of lesion (3' and 5') to create ~12nt ssDNA gap - UvrD is a helicase removing the 12 to 13 nt DNA (a single-stranded fragment is removed from the duplex) - DNA pol I and ligase fill and seal the gap

eukaryotic cells use a homologous MMR system

- 1 in 300 people carries heterozygous mutations in MMR genes (tumour suppressor gene) - mutations result in Lynch syndrome/hereditary non-polyposis colorectal cancer (HNPCC) - high risk of developing cancer - colorectal or endometrial cancer younger than age 50 think of 2 hit hypothesis for cancer: first hit - inherited mutation in one copy of an MMR gene (every cell) second hit - loss of heterozygosity + somatic mutation - these two result in inactivation of remaining WT copy of the same MMR gene (tumour precursor cell), then loss of a critical MMR activity - see replication errors and endogenous or exogenous damage in addition to MMR activity loss, which results in genetic destabilization and mutator phenotype - see mutations in APC, ras, DCC, TP53 and mutations in TGFbeta-RII which lead to cancer

transcription-coupled (NER) DNA repair

- DNA damage often blocks process of transcription - stalled RNA pol recruits nucleotide excision repair proteins, which release RNA polymerase and repair DNA - components of the general transcription factor TFIIH provide helicase activity to melt DNA at the lesion - means that genomic regions that are most highly transcribed will be subject to the greatest DNA repair in the diagram of transcription-coupled DNA repair: TOP: RNA pol transcribes DNA normally upstream of the lesion MIDDLE: upon encountering the lesion in DNA, RNA pol stalls and transcription stops BOTTOM: RNA polymerase recruits nucleotide excision repair proteins to lesion site, and then it either backs up or dissociates from DNA to allow repair proteins access to the lesion

mismatch repair system (MMR) - part 2

- MutS (MutS forms homodimer in E. coli) dimer scans DNA for distortion caused by mismatched bases in the DNA backbone (homodimer does this) - MutS embraces mismatched DNA - MutS undergoes conformation change and a DNA kink is induced (ATP is required although its exact function is unclear) - MutS: DNA complex recruits MutL (MutL is a mediator protein; not enzyme that activates and recruits MutH) - MutL activates MutH enzyme (and can move along helix to do so), which has a nicking activity - MutH nicks the one strand near the mismatched base pair, which is the orange strand - A helicase (UvrD) unwinds the DNA towards the mismatched site and an exonuclease digests the separated strand - the single-strand gap is filled with DNA pol III and sealed with a DNA ligase

summary of mutations, replication error and DNA damage

- a mutation is the result of replication errors and DNA damage - replication errors can still be significant even after proofreading - replication errors are high for repetitive sequences - DNA damage can be caused by endogenous sources (spontaneous damage and cell metabolism (ROS) and environmental factors (IR, chemicals, UV) - hydrolytic damage: depurination (AP or abasic site) - hydrolytic damage: deamination (C, A, G, 5-mC) - oxidative damage: thymine-glycol; 8-oxo-guanine - alkylation: mostly from alkylating chemicals - UV-induced dimers: CPD (TT) and 6-4 PP (TC or TT); intrastrand crossing (within same strand) - base analogs (5-bromouracil) and intercalating agents - DSBs by IR, drugs, and replication stress

more examples of alkylation

- alkylation occurs most readily at nucleophilic positions shown - highly reactive site: N3 of A and N7 of G - S-adenosylmethionine (SAM), biological methyl group donor can accidentally react w/ DNA to methylate A to give N^6-methyladenine (m^6A)

DSB repair by recombination

- double-stranded breaks are especially dangerous to cells, and if not repaired, the cell will die - homologous recombination can repair the broken DNA using homologous DNA as a template (e.g. a sister chromosome) - precise

the damaged base gets flipped out during repair

- each glycosylase is specific for a single damaged or modified base - differences between enzymes are mainly in the binding sites for the base - the damaged base (oxoG) is flipped out from the DNA by the glycosylase. the damaged base projects away from double helix where it sits in specificity pocket/catalytic site of glycosylase once it is flipped out - DNA helix is bent somewhat but adjacent base pairs are not disrupted; the distortion to its structure is modest, hence the energetic cost of base-flipping may be low.

how does the cell know which base is the misincorporated one?

- in E. coli, the Dam methylase enzyme adds methyl groups to A of GATC sequences (occurs about once every 256 bp) - after DNA replication, GATC sites are initially hemimethylated (only parental strand is marked with methylation) - Dam methylase enzyme will catch up and methylate the new strand (epigenetic modification) - before this occurs, mismatch repair system is able to distinguish the two strands as parental and newly synthesized - MutH binds to the hemimethylated site and selectively cleaves the unmethylated strand (only parental site will be marked with methylation)

mismatch repair system (MMR) in E. coli - part 1

- misincorporations are corrected by mismatch repair system - was worked out in detail in E. coli, though homologous systems exist in eukaryotes - increases accuracy of DNA synthesis by an additional 100 to 1000 times (1 error in 10^10 bases, which is considered low)

Fragile X syndrome

- most common form of inherited intellectual disability w/ a frequency of 1 in 4,000 males and 1 in 8,000 females - (CGG)n repeats in 5'-UTR of the Fragile-X syndrome gene, FMR1 (Fragile X Mental Retardation) normal = 6 - 40 carrier = 55 - 200 full expansion = 200 - 1000 (results in disease)

repair by translesion DNA synthesis

- sometimes a lesion (e.g. pyrimidine dimer or apurinic site) is not fixed before DNA replication happens - when DNA pol III encounters a lesion during DNA replication, specialized DNA pol (translesion) is recruited to synthesize DNA across the lesion (Y family of polymerases: pol IV or pol V in E. coli) steps: 1. replication complex DNA pol III with sliding clamp dissociates from DNA 2. error-prone polymerase of the Y family is recruited to polymerize across the lesion on template (upper strand) 3. the polymerase incorporates nucleotides mostly independent of base pairing (error-prone) 4. then the normal replication complex reassembles and continues 5. lesion is not fixed and mutations are produced

non-homologous end joining (NHEJ)

- template (e.g. sister chromosome) is not always available for recombination-based DSB repair - eukaryotic cells also use NHEJ to join broken ends - detection: a heterodimer of Ku70/80 binds at each broken DNA end - Ku70/80 recruits DNA-PKcs (a kinase) - other components of NHEJ are recruited: * end-processing enzymes: removal of abducts, trimming and filing; (i.e. Artemis, a nuclease) * Lig 4 * XLF and XRCC4 help bring together and align 2 ends (synapsis) - repaired ends don't always reproduce the sequence before the double-stranded break, but the cell has to repair the break or it dies

directionality of mismatch repair

- the nick by much can either be upstream or downstream of the mutation - different endonucleases are involved (exo VII or RecJ 5'->3' exo for upstream; exo I 3'->5' exo for downstream) - if DNA is cleaved on the 5' side of mismatch, then exo VII or recJ, which degrades DNA in a 5' --> 3' direction removes the stretch of DNA from the MutH-induced cut through misincorporated nucleotide - if the nick is on the 3' side of mismatch, then DNA is removed by exo I, which degrades DNA in a 3' --> 5' direction

base transition vs. transversion

- the simplest mutations are switches of 1 base for another. there are 2 kinds: transitions, which are pyrimidine-to-pyrimidine and purine-to-purine substitutions, such as T to C and A to G transversions, which are pyrimidine-to-purine and purine-to-pyrimidine substitutions such as T to G or A and A to C or T - other simple mutations are insertions of deletions of a nucleotide or a small number of nucleotides. mutations altering a single nucleotide are called point mutations.

E. coli consequences of defective NER DNA repair system

1. E. coli: - UvrABC system: main components of NER in E. coli - UvrA: recognizes DNA damage - UvrB: binds to damaged site, unwinds DNA - UvrC: cleaves damaged DNA segment; makes two cuts DNA pol I: DNA synthesis DNA ligase: ligase nucleotides removed: generally short patches (12-13 nucleotides) defects: - mutations in uvr genes lead to increased sensitivity to UV light - reduced ability to repair DNA cross-links and bulky adducts. ATP-dependent process. Recognizes damages by broad spectrum. Distortions b/c of bulky adducts and base dimers (UV-induced thymine dimers)

Human consequences of defective NER DNA repair system

2. Humans: - Main proteins: XPA, XPC, XPD, XPG, ERCC1-XPF XPC: damage recognition XPA, XPD: DNA unwinding XPG and ERCC1-XPF: make incisions DNA pol epsilon or gamma: DNA synthesis DNA ligase I: ligase nucleotides removed: longer patches (~30 nucleotides) defects lead to genetic disorders, such as: - xeroderma pigmentosum (XP): increased sensitivity to UV light and high risk of skin cancers. caused by mutations in genes (XPA to XPG). - trichothiodystrophy (TTD): photosensitivity, brittle hair and nails, and intellectual disability. associated with mutations in genes involve in NER, especially XPB or XPD (subunits of TFIIH) ATP-dependent process. Recognizes damages by broad spectrum. Distortions b/c of bulky adducts and base dimers (UV-induced thymine dimers)

base excision repair (BER) differs from nucleotide excision repair (NER) in which of the following ways? A) BER recognizes helix distortions, while NER recognizes specific base damage B) NER recognizes helix distortions, while BER recognizes specific base damage C) BER involves a DNA synthesis step, while NER does not D) NER involves removal and replacement of DNA containing altered bases, while BER does not

B) NER recognizes helix distortions, while BER recognizes specific base damage Base excision repair (BER) is specialized for fixing non-bulky, small-scale damage to individual bases, such as those caused by deamination or oxidation. It typically recognizes specific altered or incorrect bases without major distortion to the DNA helix. Nucleotide excision repair (NER), on the other hand, is responsible for removing bulky lesions, such as pyrimidine dimers caused by UV radiation that distort the DNA double helix. Both BER and NER involve a DNA synthesis step to replace removed nucleotides and both involve removal and replacement of damaged DNA sections

Dam methylase helps mismatch repair (mr) in E. coli. Which of the following is true if Dam methylase is absent? A) no effect on mismatch repair (mr) B) newly synthesized daughter strand containing incorrect base and parental strand will both be unmethylated SO either strand serves as template strand for mr C) all DNA would remain methylated SO either strand serves as template strand for mr D) only parental strand would be methylated SO only parental strand serves as template strand for mr

B) newly synthesized daughter strand containing incorrect base and parental strand will both be unmethylated and, therefore, either strand serves as template strand for mismatch repair in E. coli, the mismatch repair system distinguishes the newly synthesized strand from the parental strand by the methylation status of the DNA. The parental strand is methylated by Dam methylase at adenine residues within the sequence GATC. Immediately after replication, the new strand is not yet methylated, which allows repair system to target unmethylated strand for correction. If Dam methylase is absent, both strands will remain unmethylated, and the mismatch repair system loses its strand discrimination capability, theoretically allowing either strand to be used as the template. this could lead to errors because the system could not reliably identify and correct mismatches on the newly synthesized strand

methylation and subsequent deamination of cytosine produces what type of mutation after one round of DNA replication? A. C-to-T transversion B. C-to-T transition C. G-to-A transition D. C-to-A transversion

B. C-to-T transition the methylation of cytosine followed by deamination typically results in the conversion of cytosine to thymine. after one round of DNA replication, this would lead to a C-to-T mutation on the original DNA strand where the alteration occurred, and a G-to-A mutation on the complementary strand, b/c the DNA polymerase would insert an adenine opposite the altered base, which it would now interpret as a thymine. the correct answer is B. C-to-T transition this is b/c a transition mutation refers to a purine being replaced by another purine, or a pyrimidine being replaced by another pyrimidine, and in this case cytosine (pyrimidine) is replaced by thymine (also pyrimidine).

given type of damage caused by cisplatin, which DNA repair pathway is most suited to address intrastrand crosslinks induced by this drug? why can't BER or TLS work for this scenario?

BER is meant to fix small, non-helix-distorting lesions on DNA, such as those caused by oxidation, alkylation, or deamination of individual bases. BER enzymes recognize specific damaged/inappropriate bases, remove them, and repair the single nucleotide gap. BER not equipped to handle the large, helix-distorting damage cisplatin-induced crosslinks consist of. TLS allows DNA replication to bypass lesions that would stall the replication fork, using polymerases that could synthesis DNA across damaged areas. But TLS doesn't remove the lesions and allows the cell to replicate DNA despite their presence, possibly leading to mutations. Bypassing lesions w/o removing them in the case of the cisplatin-induced damage could preserve the crosslinks and risk interference with essential DNA functions and cell viability. NER directly addresses the issue by recognizing, removing, and repairing bulky, helix-distorting damage, making it most suitable for cisplatin-induced intrastrand crosslinks.

translesion DNA synthesis accomplishes which of the following? A) repairs DNA mutations B) removes damaged DNA C) allows DNA replication to proceed around DNA lesions that stall the replication fork D) stalls the replication fork

C) allows DNA replication to proceed around DNA lesions that stall the replication fork translesion DNA synthesis (TLS) is a DNA damage tolerance process allowing DNA replication machinery to replicate past DNA lesions such as thymine dimers or bulky chemical adducts that would otherwise block the progression of the replication fork. specialized TLS polymerases, which can accommodate distorted DNA structures in their active sites, are involved in this process. these polymerases can insert nucleotides opposite the damaged bases, allowing replication to continue, albeit often with a higher chance of introducing mutations due to their lower fidelity compared to regular DNA polymerases.

which one of the following statements is true regarding transcription-coupled repair? A) transcription-coupled repair corrects damage in mRNA B) transcription-coupled repair occurs only during S phase of the cell cycle C) transcription-coupled repair involves all the major nucleotide excision repair proteins, as well as RNA polymerase D) transcription-coupled repair occurs in response to stalling of a ribosome

C) transcription-coupled repair involves all the major nucleotide excision repair proteins, as well as RNA polymerase transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that specifically targets lesions causing stalling of RNA polymerase during transcription. this mechanism ensures that the transcribed strand of active genes is quickly repaired, which is crucial for the maintenance of genetic integrity and the prevention of mutations that could result from transcription-blocking lesions TCR involves typical NER factors as well as additional components that recognize stalled RNA polymerase and recruit repair machinery

Q5. Describe the types of DNA damage and their repair mechanisms, including crosslinks, oxidative damage, AP sites, and double-strand breaks (part 2)

Crosslinks: Interstrand crosslinks are complex and need a combo of NER, translesion DNA synthesis (TLS) and homologous recombination (HR) to repair both strands. Oxidative Damage: Oxidative base damages like 8-oxoguanine are targeted by BER w/ glycosylases like 8-oxoguanine DNA glycosylase (OGG1), which recognizes and removes damaged bases. AP Sites: Created spontaneously or by BER initiation, apurinic/apyrimidinic sites (AP sites) are processed by AP endonucleases that make a nick in DNA, and gap is filled by DNA pol I in E. coli (or Pol β in eukaryotes) and sealed by DNA ligase. Double-Strand Breaks (DSBs): Repaired by nonhomologous end joining (NHEJ); quickly rejoins broken ends but can cause loss or gain of nucleotides at the repair site, or by homologous recombination (HR), which is an error-free repair mechanism using sister chromatid as a repair template, mostly during S and G2 cell cycle phaes. Vital for correcting DNA damage and ensures cell stability and survival.

which one of the following is an example of a mutation? A. oxidation of guanine to produce 7,8-dihydro-8-oxoguanine or oxoG B. formation of a thymine dimer C. alkylation of guanine to produce O^6-methylguanine D. deamination of cytosine, followed by a round of DNA replication

D. deamination of cytosine, followed by a round of DNA replication Each of the listed options (A, B, C, D) describes a type of DNA damage, but not all of them would result in a mutation unless they are perpetuated through a round of DNA replication or are not repaired prior to DNA replication option D is the only option that explicitly states the occurrence of DNA replication after the damage. Deamination of cytosine converts it into uracil, which could base-pair with adenine. Upon replication, it can result in a C-G to T-A transition mutation. If one considers the criteria for something to be classified as a mutation, which is a change in the DNA sequence that is passed on to the next generation of cells, option D is the most direct example of a mutation, as it includes the replication step that would fix the change into the genome.

Q1. Differentiate between DNA damage and mutation.

DNA damage refers to physical abnormalities in DNA, such as strand breaks, missing bases, or chemically modified bases caused by various environmental factors like UV light, radiation, or chemical agents. DNA damage is often recognized and repaired by cellular mechanisms to restore the original DNA sequence, Mutations are a permanent alteration in the DNA sequence making up a gene. Mutations can occur if DNA damage is not repaired before DNA replication or if it is replicated or misrepaired in a way that changes the original sequence. While DNA damage could potentially lead to a mutation, it is not a mutation itself until it is fixed into the DNA sequence and propagated to successive generations of cells.

Q6. Contrast the repair processes MMR, BER, NER, TLS, and NHEJ vs. DSBR by HR (part 1) - Mechanism of MMR in E. coli; The role of methylation - Mechanism of BER and NER.

Mismatch Repair (MMR) in E. coli + role of methylation: MMR corrects errors escaping proofreading by DNA pol, like misincorporated bases and small insertion-deletion loops. MutS protein detects mistmatch; MutL binds and recruits MutH, which cleaves unmethylated daughter strand. Section of DNA containing the error is removed, and DNA pol III resynthesizes correct sequence. Methylation of parent strand guides system to correct newly synthesized, unmethylated DNA. Base Excision Repair (BER) mechanism: BER fixes small, non-distorting base lesions. DNA glycosylases recognize and remove damaged bases, creating an AP site. AP endonuclease cleaves phosphodiester bond, and DNA pol I fills gap while DNA ligase seals it. Nucleotide Excision Repair (NER) mechanism: NER removes bulky, helix-distorting lesions. Uvr proteins in E. coli recognize distortions, excise a short single-stranded DNA segment around the lesion, and DNA pol I synthesizes new DNA to fill gap that is then sealed by DNA ligase.

Q5. Describe the types of DNA damage and their repair mechanisms, including single nucleotide variations, deamination, bulky DNA lesions and T-T dimers (part 1)

Single Nucleotide Variations (SNVs): Repaired predominantly by mismatch repair (MMR), which scans DNA immediately after replication for mispaired bases, removes the incorrect base, and then uses the parent strand as a template to insert the correct nucleotide. Deamination: Base excision repair (BER) identifies and excises the altered base (e.g., uracil from deaminated cytosine), then fills in gap w/ correct base using short-patch repair synthesis Bulky DNA Lesions: Nucleotide excision repair (NER) recognizes these distortions and removes a short single-stranded DNA segment encompassing the lesion, w/ DNA polymerase and DNA ligase filling and sealing the gap, respectively. Thymine Dimers: Specific types of bulky lesions caused by UV radiation and are repaired by the NER pathway, which removes the dimer along with a few nucleotides on either side.

Q3. What is a tautomeric shift and how does it lead to mispairing during DNA replication?

Tautomeric shift is where a chemical rearrangement in which a base undergoes a proton shift and has its bonding structure altered. This results in alternate forms, or tautomers or the base that can lead to atypical base pairing during DNA replication. KETO FORM: cytosine (C) pairs w/ guanine (G) or thymine (T) pairs w/ adenine (A) ENOL FORM: cytosine (C) pairs w/ adenine (A) or thymine (T) pairs w/ guanine (G) if tautomeric shift occurs in a base just before replication, the misfiring can become permanent, resulting in a point mutation -- a transition or transversion depending on the new pairing.

abasic site can also cause _______ breakage

abasic site can also cause strand breakage - at an abasic site, ribose 'open' form is in equilibrium w/ the 'closed' form - in 'open' aldehyde form, the 3'-phospodiester bond is unstable; therefore, hydrolysis of the phosphodiester bond at the 5' site may result in strand break.

base excision repair (BER)

e.g. uracil is formed by deamination of cytosine (hydrolytic damage) - uracil glycosylase scans (diffuses along minor groove of DNA) for uracil - glycosidic bond is hydrolyzed/broken by uracil glycosylase, releasing uracil and leaving an AP site (apurinic/apyrimidinic site) to remove damaged base - resulting abasic sugar is removed from DNA backbone in a further endonucleolytic step - endonucleolytic cleavage also removes apurinic and apyrimidinic sugars arising from spontaneous hydrolysis - AP endonuclease cleaves DNA backbone at the 5' position, leaving a 3' OH - an exonuclease cleaves DNA backbone at 3' position, leaving a 5' phosphate - DNA polymerase I and ligase re-synthesize to fill gap and seal the strand after the damaged nucleotide has been fully removed from the backbone

4. action of intercalating agents

intercalating agents: flat molecules containing polycyclic rings that can bind to/slip between equally flat purine or pyrimidine bases of DNA - e.g., proflavin, acridine orange, ethidium bromide - cause deletion or addition of base pairs

DNA damage repair and tolerance systems - summarized

mismatch repair damage: replication errors enzymes: MutS, MutL, and MutH in E. coli; MSH, MLH, and PMS in humans photoreactivation damage: pyrimidine dimers enzymes: DNA photolyase base excision repair damage: damaged base enzymes: DNA glycosylase nucleotide excision repair damage: pyrimidine dimer; bulky adduct on base enzymes: UvrA, UvrB, UvrC, and UvrD in E. coli; XPC, XPA, XPD, ERCCI-XPF, and XPG in humans double-strand break repair damage: double-strand breaks enzymes: RecA and RecBCD in E. coli translesion DNA synthesis damage: pyrimidine dimer, apurinic site, or bulky adduct on base enzymes: Y-family DNA polymerases, such as UmuC in E. coli

name the proteins in each step of the DNA repair pathways in E. coli (recognition, excision, DNA synthesis, ligation) for MMR, BER, and NER

mismatch repair (MMR) recognition: MutS, MUtL excision: MutH, exonuclease I (exo I), exonuclease X (exo X) and exonuclease VII (exo VII) DNA synthesis: DNA pol III ligation: DNA ligase base excision repair (BER) recognition: DNA glycosylases (e.g. uracil DNA glycosylase for uracil) excision: AP endonuclease (endo IV), deoxyribose phosphodiesterase (for removing sugar phosphate) DNA synthesis: DNA pol I ligation: DNA ligase nucleotide excision repair (NER): recognition: UvrABC complex (UvrA, UvrB, UvrC) excision: UvrC (incision), UvrD (helicase for strand removal) DNA synthesis: DNA pol I ligation: DNA ligase *** these proteins work in sequence where first the damage is recognized, then the incorrect or damaged nucleotide is excised, followed by the synthesis of new DNA to replace the excised section, and finally, the newly synthesized DNA is joined to the existing strand by DNA ligase

cisplatin is a chemotherapy drug widely used to treat various cancers. it binds DNA and induces intrastrand crosslinks. the crosslinks create bulky lesions that distort DNA helix, leading to replication and transcription errors. their accumulation and disruption of cellular processes can cause cell death, so cisplatin is effective against cancer cells. given type of damage caused by cisplatin, which DNA repair pathway is most suited to address intrastrand crosslinks induced by this drug?

the type of damage caused by cisplatin, which are intrastrand crosslinks that create bulky lesions and distort the DNA helix, is most suitably addressed by the nucleotide excision repair (NER) pathway. NER is specialized for removing a wide range of helix-distorting lesions, including bulky chemical adducts and intrastrand crosslinks, by excising a short single-stranded DNA segment that contains the lesion and then fills in gaps with newly synthesized DNA.

DNA damage tolerance: survival and mutagenesis

when it comes to cellular defences against DNA damage.... cells use DNA repair pathways to restore DNA to undamaged state. if DNA damage is present when the genome is being replicated, the cell must use DNA damage tolerance to avoid a block in replication and a potentially lethal double-strand break. translesion DNA synthesis replicates across DNA lesion, but the lesion remains in the genome until a DNA repair pathway can subsequently correct the damage