Biochem - DNA Repair Flashcards by Stephen a

DNA proofreading is

3’–>5’ exonuclease activity

Not perfect

How well did you know this?

Not at all

Perfectly

Agent –> Damage

Spontaneous rxn, reactive oxygen, alkylating agents

Modified bases, abasic sites, ssDNA breaks

How well did you know this?

Not at all

Perfectly

Agent –> Damage

UV light, polycyclic aromatic hydrocarbons

Pyrimidine dimers, bulky adducts

How well did you know this?

Not at all

Perfectly

Agent –> Damage

Xray, antitumor Cisplatin

DNA insterstrand cross links, dsDNA breaks

How well did you know this?

Not at all

Perfectly

Deamination
Hypoxanthine
Xanthine

C –> A
A –> H
G–> X

How well did you know this?

Not at all

Perfectly

UV light

thymine pyrimidine dimers between neighboring pyrimidines

How well did you know this?

Not at all

Perfectly

Reactive oxygen species ROS

oxidize DNA

G –> 8-oxoguanine

How well did you know this?

Not at all

Perfectly

Alkylating agents

Methylate DNA

How well did you know this?

Not at all

Perfectly

of DNA lesions per cell per day

100,000

How well did you know this?

Not at all

Perfectly

Mutations

Hereditary changes in DNA
Permanent changes that can be passed down to offspring
Germline –> passed to offspring
Somatic –> lead to cancer

How well did you know this?

Not at all

Perfectly

Major categories of mutations

Point mutations in single bp changes
Affecting copy number of small repeats (duplications, di tri NT repeat expansion
Large insertions/deletions
Chromosomal rearrangements - translocations
Chromosomal abberations - loss or gain of chromosomes or large parts

How well did you know this?

Not at all

Perfectly

Two DNA protection strategies

Damage avoidance

DNA repair

How well did you know this?

Not at all

Perfectly

Damage avoidance

First line of defense
1 - inactivate ROS w/reducing agents or SOD
2 - Shield organisms from radiation (melanin in skin)

How well did you know this?

Not at all

Perfectly

Superoxide dismutase

Reduces O2 –> H2O2
Catalase H2O2 –> H20
Most important in mitochondria - where ROS are made

How well did you know this?

Not at all

Perfectly

DNA repair

MMR
BER
NER
HR
NHEJ

How well did you know this?

Not at all

Perfectly

Three R’s of DNA repair

1 - Damage is recognized
2 - Damage is removed
3 - Single stranded gap is replaced by DNA pol
4 - Newly synthesized DNA is ligated

How well did you know this?

Not at all

Perfectly

DNA synthesis can occur outside of ___ phase

S phase

Can happen in different parts

DNA Mismatch Repair MMR

Repairs errors that were missed by proofreading
Acts within very short time window after DNA replication
1 error / 1 billion BP

E. Coli MMR

1 - parent strand is recognized
2- Mut proteins are called upon
3 - MutS recognizes and binds damaged DNA
4 -MutL joins to promote a loop structure
5 - MutH recognizes the methylated GATC and has endonuclease activity to make a nick
6 - MutH removes dNMPs in region around it
7 - Resynthesis is carried out by DNA pol III and ligase

How is parent strand recognized in E. coli?

Parent is methylated at GATC sequence by DNA adenine methylation methylase
Methylation is absent in the newly synthesized DNA just after replication

MMR comparison in eukaryotes

Several MutS/MutL homologs

No MutH, Dam - strand discrimination is unknown

MMR Deficiency in eukaryotes

responsible for microsatellite di nucleotide instability
leads to hereditary non-polyposis colorectal cancer (HNPCC)
Autosomal dominant - polyps progress to cancer quickly

Base excision repair (BER)

Repair of modified bases and uracil misincorporations

Acts at any cell cycle

BER process

Modified base is cleaved by a specialized DNA glycosylase
Apurinic/apyramidic site formed by AP endo/exonuclease (lyase)
One NT gap is filled by DNA pol beta
Ligase seals gap

Nucleotide excision repair NER

Acts at any cell stage Repairs any large bulky lesions in DNA Pyrimidine dimers, bases modified by covalent binding of polycyclic aromatic hydrocarbons PAH

NER Process

1 - DNA damage is recognized 2 - Helicase unwinds the dsDNA 3 - Excinuclease binds and cleaves damaged DNA at both sides of lesion 4 - Removal of ssDNA fragment 30nt 5 - Gap filled by DNA pol (replicative or DNA pol kappa) and ligase

Diff between human and prok excinucleases

Human - 30nt | Prok - 12nt

Xeroderm pigmentosum

Key role in NER Caused by defects in any of seven proteins involved in NER for humans Skin cancer at rate of 2000x normal - extreme photosensitivity

Transcription coupled NER TC-NER

specifically repairs DNA damage in actively transcribed genes ERCC6/8 Mutation --> Cockayne syndrome

NER

Damaged bases removed as oligonucleotides Removal of UV induced damage, 20% of oxidative damage Incision on both sides of lesion - excinuclease Deficient in human disorders

BER

Damaged bases removed as free bases Removal of oxidative and alkylation damage Incision on one side of lesion Role in aging and cancer

Homologous Recombination HR

Error free Uses homologous dsDNA as a template Can only function when sister chromatid (mitosis) or homologous chromosome (meiosis) are available Only function in late S phase and G2 of cell cycle

Major threat to cell survival?

dsDNA breaks Single unrepaired DSB is lethal for cell X-rays, cross linking chemical agents, endogenous free racidals hit DNA in both strands

HR process

1 - dsDNA break 2 - 5-->3' resection by exonuclease 3 - RAD51 homolgous pairing protein loads onto ssDNA to search for homolgous template 4 - Template is used for DNA synthesis 5 - 3' end acts as primer 6 - resynthesis of other strand using newly made strand as template 7 - ligation

Non-Homologous End Joining NHEJ

repairs DSB in template independent manner Error prone Non-random facilitated ligation of broken DNA ends DNA loss is irreversible Can be used at any stage of cell cycle

NHEJ process

1 - dsDNA break 2 - End joining by KU70/80 and DNA-PKcs 3 - End trimming by exonucleases make the ends match up better 4 - ligation

Deficiencies in HR

ATM - checkpoint kinase

Damage Tolerance

Last resort Use bypass polymerases with reduced accuracy DNA pol dissociate from fork and bypass DNA pols attach Error prone - responsible for damage induced mutations POL IV, POL V in proks

Bypass DNA polymerase

Lack 3-->5' proofreading | Distributive rather than processive polymerization

Error free/prone

Error Free: MMR, BER, NER, HR Error Prone : NHEJ, Lesion bypass