L211 - DNA Damage

trinucleotide repeat disorder

3 bases repeated over and over again Ex. CAG in Huntington's Disease

alkylation

Add an alkyl group to a nucleotide (add methyl, ethyl, etc) Alkylation of guanine mispairs with thymine; transition

mutagen

Agents that generate mutation inducing DNA damage -Chemical (Nitrous acid, mustard gas) -Environmental (Radiation: UV, ionizing) -Biological Can lead to -Chemical modification of the bases -Formation of nucleotide dimers -Formation of double-stranded breaks

What features do mismatch repair, base-excision repair, and nucleotide-excision repair have in common?

All correct DNA damage; recruit proteins for repair mismatch and BER removes incorrect bases

Compare and contrast base excision repair and nucleotide excision repair. Diagram both processes. Provide examples of the types of damage each is primarily responsible?

BER - damaged bases ex. Mismatch, abasic sites NER - errors in shape -T-T dimers, bulky adducts

N-glycolyases

BER - works on glycosidic bond to remove bases leaving an abasic site: apurinic or apyrimdinic

AP-endonuclease

BER: hydrolyzes the phosphodiester bond

A nick in two strands fragment both daughter chromosomes

Both daughter chromosomes broken

Double Stranded Break

Caused by -Ionizing radiations (X rays & gamma rays) -Exposure to exogenous agents -Replication fork collapse during repair -Superoxide reactions

Error prone repair

DNA repair that leads to a loss of sequence -major component of mutagenesis Ex. NHEJ, Translesion synthesis

base-flipping

Damaged base is flipped away from the double helix to expose it in order to cleave it during BER

nucleotide excision repair

Detects error in the shape of the DNA helix -Ex. Thymine-thymine dimers, bulky adducts Bacterial Pathway UvrA and UvrB proteins act as damage sensor --> UvrB (helicase) unwinds the damaged region --> UvrA released UvrC comes in and makes 2 cuts on either side of the damage (exinuclease) UvrD (helicase) causes strand displacement--> UvrC released Repaired by DNA pol I or DNA pol III, sealed by ligase

mutation

Different sequence than the normal/standard/functional sequence -structurally represent DNA -inherited by daughter molecule [Changes take place in germ cells-‐‐ the gametes-‐‐ eggs and sperm]

hemimethylated

During DNA synthesis, methyl groups are only on the template strands -E. coli

mismatch repair

E.coli MutS dimer scans DNA --> Recognizes distortion --> changes conformation with ATP MutL recruited --> MutL-MutS dimer activates MutH -MutH associates with hemimethylated sites on template -When MutL-MutS dimer reaches the nearest MutH (closest unmethylated GATC sites), endonuclease capacity activated -Helicase and exonucleases create a gap all the way from nick to past mismatch Helicase (UvrD) unwinds nicked strand --> An exonuclease removes DNA from the nick to the distortion DAM methylase methylates A nucleotide at 5'-GATC-3' sequence DNA pol fills in the gap, sealed with ligase *discrimination between template strand & newly synthesized strand b/c template is methylated

Cancer therapy

Ionizing radiation - DSBs Cisplatin induces interstrand crosslinking Etoposide is topoisomerase inhibitor that creates strand breaks Others are DNA alkylating agents

deamination

Loss of an amino group from a nucleotide base -Deamination of cytosine converted to uracil CG-->TA; Transition mutation Ex. Nitrous Acid

depurination

Loss of one of the purines (adenine & guanine) from a nucleotide -One of the most frequent spontaneous chemical changes affecting DNA GC-->AT; Transition mutation

Contrast the various types of DNA repair mechanisms known to counteract the effects of UV radiation. What is the role of visible light in repairing UV-induced mutations?

Mismatch repair vs. DNA photolyase DNA photolyase absorbs visible light as energy which is used to break T-T dimers

reactive oxygen species

Molecules that contain oxygen -superoxide, NaOCl, H2O2 *Cause DSBs

Mismatch repair in Eukaryotes

Not clear how old and new DNA strands identified -Directed by presence of ssDNA at strand breaks -at the 3' end of the leading strand or at the ends of Okazaki fragments

Name two enzymes that catalyze the direct reversal of DNA damage? Outline their mode of action

O6-methylguanine methyltransferase reverses methylation of O6 of guanine -Suicide enzyme (enzyme can't preform another rxn) DNA photolyase repairs damage caused by exposure to ultraviolet light -absorbs light energy and uses it to break T-T bond

A nick in one strand fragments one chromosome

One daughter chromosome broken

Oxidation

Oxidation of guanine pairs with adenine -Transversion

Synthesis Dependent Strand Annealing

Repairs DSBs Invading strand extended by DNA synthesis -Complementary strands used as templates -Synthesis continues until new DNA can pair with 3' overhand

Holliday Junction

Resolvases carry out cleavage at junctions Occurs during Meiosis - homologous recombination Resolved by cleavage of DNA at one of two sites *Holliday Junction resolution determines whether recombination occurs*

How do glycolysases gain access to these damaged bases to cleave it in BER?

Scans DNA until it detects a specific lesion Damaged base is flipped away from the double helix to expose it in order to cleave it

Transition coupled NER

Special mechanism to preferentially repair damage in genes that are actively being transcribed RNA pol transcribes DNA into mRNA --> RNA pol stalls at distortion --> recruits NER to site

Resistance to Chemotherapy

Targeting the principal DNA damage response actors promotes cell death to limit cancer progression Chemo is DNA damage inducing agent: cause rapidly proliferating cell to die by overwhelming damage Tumors accumulate more mutations: one way they become resistant to drugs --> UPREGULATE a particular DNA damage pathway

How does transcription-coupled NER differ from global genome NER?

Transcription coupled occurs while gene is actively being transcribed; specific Global genome NER can happen ANYWHERE on genome

DNA pol IV, V

Translesion synthesis polymerases that helps cells survive derailment; cross lesions to extend primer strand even when template is damaged -E. coli

Homologous Recombination

Typically the principal pathway used to repair DSB during replication Homologous chromosomes provide template for repair

pyrimidine dimer (thymine dimer)

UV induced damage -cross linking of adjacent thymine residues

Non-homologus end joining (NHEJ)

Usually occurs when cells have not yet duplicated DNA (G0 and G1) Joins broken ends of DNA together Major pathway in multicellular eukaryotes DSB --> resection to expose ssDNA --> alignment of region at microhomology --> trimming of overhands (loss of sequence - mutagenic) --> Ligation Ku70 & Ku80 *Error prone repair

Translesion Synthesis

When replicative DNA polymerases hit DNA lesion such as abasic sites or T-T dimers, they can be derailed -E. coli

Nonsense mutation

creates a completely nonfunctional protein

Exinuclease

cuts out a fragment by hydrolyzing two phosphodiester bonds, one on either side of the lesion in the DNA

germline mutation

detectable and heritable variation in the lineage of germ cell -transmitted to offspring

MutS

dimer that scans DNA; recognizes distortion; changes conformation with ATP -E. coli

Ku70 & Ku80

form heterodimer -binds broken DNA ends keeps them in close proximity -Recruits additional proteins (include nucleases that process tails; ligase to join ends) Ends brought together by microhomology; ligation

MutL

forms dimer with MutS to activate MutH

MutH

generates a single stranded nick in the newly synthesized strand; recognizes the nicks and non-methylated strand -E. coli

Silent mutation

has no effect on amino acid produces

Xeroderma pigmentosum

human disease caused by mutations in any one of several nucleotide excision repair genes -recessive disease resulting in hypersensitivity to sunlight & UV -skin disorders & cancer predisposition

What does superoxide react with?

macromolecules; main target is DNA; can chemically modify bases or cause DSBs

UvrC

makes a single-stranded nick on BOTH sides of the distortion

photoreactivation

mechanism for repairing damage by DNA photolyase

DAM methylase

methylates DNA on A nucleotide at 5'-‐‐GATC-‐‐3' sequences

induced mutation

mutations caused by exogenous agents (mutagens)

DSB repair by Recombination

needs second intact dsDNA (sister chromatid) Resection + strand invasion --> forms D loop Synthesis Dependent Strand Annealing

insertions

one or more nucleotides added to the DNA sequence

deletions

one or more nucleotides removed from the DNA sequence

Frameshift mutation

order of nucleotide sequence changes -insertions & deletions

transitions

point mutation that changes a purine nucleotide to another purine (A ↔ G) or a pyrimidine nucleotide to another pyrimidine (C ↔ T)

polymerase slippage

polymerase falls off trinucleotide repeats which can form secondary hairpin structures -When the polymerase reattaches, it begins copying earlier than where it left off (because the hairpin makes the structure appear shorter) so there are more repeats

fail safe glycolyases

recognizes G:A pair -occurs when guanine is oxidized, pairs with A BER

UvrD

removes the single-stranded fragment; DNA polymerase and ligase repair and seal the gap

DNA photolyase

repairs damage caused by exposure to ultraviolet light -absorbs light energy and uses it to break T-T bond

DNA Damage Response

response system consisting of many proteins that form a signaling network -DNA damage sensing (proteins that are recruited directing to the site of damage) -Signaling -Activation of appropriate response

UvrA

scans DNA and recognizes distortions

What genetic defects result in the disorder xeroderma pigmentosum (XP) in humans? How do these defects create the phenotypes associated with the disorder?

Mutation in NER pathway NER can't repair UV damage, so skin is sensitive & damage may cause cancer

Spontaneous Mutations

Mutations due to errors during DNA replicated (endogenous agents)

If an oxidative lesion occurs spontaneous in a ssDNA fragment generated on the lagging strand during replication, it is not readily repaired by NER or BER. Why?

NER & BER occur only in dsDNA. Both processes excise the damaged base(s) from the damaged strand, leaving a gap that can be filled in -- but only if an undamaged complementary strand is present

Global genome NER

NER on several types of damage and anywhere in genome

What happens if DAM methylase is absent?

The new synthesized daughter strand containing the incorrect base and the parental strand will both be unmethylated and therefore either strand can serve as the template strand for mismatch repair

Resection

a nuclease digests a short stretch of DNA, creates ssDNA with 3' tails

apurinic (AP) site

abasic site location in DNA that has neither a purine nor a pyrimidine base, either spontaneously or due to DNA damage

Microhomology

brings ends together of NHEJ

point mutations

causes a single nucleotide base substitution, insertion, or deletion of the genetic material

base-pair substitution mutation

causes a single nucleotide base substitution, insertion, or deletion of the genetic material -point mutation

Missense mutation

change one nucleotide which resulting in coding for a different amino acid

Base excision repair

corrects damaged bases -Recognizes specific chemical modifications on a SINGLE BASE within the DNA structure Mechanism: 1. Recognition and removal of damaged base by DNA glycosylase 2. Recognition of abasic site by AP endonuclease 3. Cleavage of phosphodiester bonds flanking abasic site 4. Replacement of excised bases by DNA polymerase + ligase Corrects for -presence of Uracil in DNA (due to deamination of C) -deamination -methylated bases -oxidized bases

O6-methylguanine methyltransferase

reverses methylation of O6 of guanine -Suicide enzyme (enzyme can't preform another rxn)

UvrB

scans DNA and recognizes distortions; creates a bubble around the distortion after UvrA leaves

Strand invasion

ssDNA tail invades intact duplex and base pairs to make heteroduplex

transversions

substitution of a (two ring) purine (A & G) for a (one ring) pyrimidine (C & T) or vice versa

Complement Analysis

way of distinguishing mutations in the same gene from mutations in different genes (that are involved in the same pathway) 1. If mutations are in separate genes, F1 will be heterozygous at both loci 2. If mutations are in same gene F1 is homozygous for 2 mutant alleles