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