Genotoxic stress Flashcards
What is Genotoxic stress
Refers to the insult or deleterious effect or biological, chemical or physical agents on the hereditary material and releated genetic functions
What is involved in mutagenesis
Damage to cellular DNA and the development of cancer
Can genomic mutations be carried over into the daughter generations of cells
YES if the mutation is not repaired prior to mitosis
What are the three possible responses once cells lose their ability to repair damaged DNA
Senescent (irreversibly dormant), apoptotic, or maligant
What are examples of exogenous genotoxic agents
Chemical or physical nature such as chemical mutagens UV light or ionizing radiation
What are examples of endogenous factors
Regular products of cellular metabolism ROS and ordinary errors in DNA replication
What are modfications of indvidual nucleotides
Demination, depurination, oxidation, alkylation
What is exogenous DNA damage usually associated with
Bulky lesions and ss/ds
What is the phosphate group linked by
Phosphoester bond to a pentose sugar which is then linked to a N-glycosyl bond
What are the three sensitivie sites for DNA lesions
Amino group of adenine and cytosine
N-glycosyl bonds
Phosphodiester bonds
What is depurination and does it occur spontaneously for mismatch excision repair
Hydrolysis of N-glycosyl bond occurs spontaneously when the base is purine
What happens in depurnation
Releass guanine and adenine resulting in abasic/apurnic site
What is Demination
Spontaneous removal of an amino group from cytosine guanine and adenine which coverts it to uracil xanthine and hypoxanthine
What is 8-Oxoguanine
Oxidation of guanine where guanine normally pairs with cytosine 8-oxoguanine can also mis pair with adiene
What is the substitution called from 8-oxoguanine
CG to AT
What is the most common type of point mutation in humans
C to T which is caused by demination of 5-methyl-Cytosine
How are T-G mismatches always repaired
Removing the T and replacing it with aC which is called base excision repair
WHat does base excision repair take place
Prior to DNA replication
What are the four major steps of BER
- Recognition of damaged bases and T-G mismatches by DNA glycosylase which flips the base out of the helix and hydrolyzes the N-glycosidic bond leaving AP site
- DNA strand cut; AP endonuclease cuts the DNA backbone at the AP site
- Excision of the AP site; AP lyase which is associated with DNA polymerase B removes the dexoyribose-phosphate AP site making a gap within DNA strand
- Gap repair DNA polymerase B fills the fap and DNA ligase seals it
When is mismatch excision repair used
Base-pair mismatches and insertion or deletions of one or few nucleotides that are accidentally introduced by DNA polymerases during replication
What are glycosylases sensors to
Damaged bases and mismatches
What are the three steps of mismatch repair
- Recognition of mispaired segment of DNA by MSH2/MSH6 protien complex which distinguishes between the template and newly synthesized daughter strand
- Excision of DNA segment around the mismatch
- Gap repair DNA polymerase S fills the gap and DNA ligase connects the new section of DNA to the backbone
What are the three different types of DNA-modifying enzymes for Step 2
For mismatch excision repair
DNA endonuclease complex (MLH1 and PMS2) which cuts the newly synthesized daughter strand DNA helicase unwinds the helix and DNA exonuclease which removes the nucleotides from the cut ends including mismatched base
What is MSH2 and MSH6
A sensor
What is Lynch syndrome associated with
Mutant forms of protiens essential for mismatch exicsion repair
What is a predispostion of nonpolyposis colorectal cancer
Loss of function mutations of the sensor protien MSH2 and MLH1
What does alkylating agents do
Transfer carbo-containg methy and ethyl to N and O atoms in the bases which then particpate in hydrogen bonds in DNA double helixx
What can the modfication of alkylating agents do
Innocuous cytotoxic or mutagenic
What alkylation is harmless
N7 of guanine does not change the base=paring properites
What alkylation causes harm
N3 of adenine is more serious because it creates a base that cannot base pair properly with any other base a non coding base DNA replication will then then stop and kill the cell and the base modification is cytotoxic
What do mutagenic mutations result from
Alkylation of the nitrogen and oxygen atoms that are directly involved in base paring
What is an example of a mutagenic mutation
Alkylation of the O6 guanine by EMS which transfers the ethyl group to DNA leads to the replacment of. G-C pair by A-T pair. O6 ethylguanine base pairs with T instead of cytoside
What is EMS used
Common laboratory mutagen which is used to induce mutations in different types of cells
What is mustard gas from
Alkylating agents have two recative sites which creat interstrand crosslinks due to cytoxic effects
What is alkyltransferases
Capable of re-pairing DNA by damage reversal by removing methyl, ethyl and even larger alkyl groups
How do alkyltransferase work
Single step reaction direct alkyl group molecule from DNA to a cysteine residue (sulfhydryl group) in the active center of the repair molecule. Alkyltransferase then becomes irreversibly inactivated and guanine is restoresed
Where does the alkyltransferase remove alkyl groups from what positions
O6 from guanine and O4 poition of thymine
What is alkyltransferases known as
Sucide enzyme because it is used up during the repair reaction
Do all bases asborb UV light
YES
What does UV damage do
Cross links adjacent pyrimidines on the same DNA strand
What is the most common type of DNA damage and what is refered to as
Formation of thymine-thymine dimers connected by C-C bonds known as bulky lesions because the normal DNA shape is distoreted making DNA Kinks
What does nucleotide excision repair fix
DNA regions containing chemically modified bases or bulky damage that distort the normal shape of DNA locally
What are the four steps of NER
- Initial recognition of pyrimidine dimers by a XP-C 23B protien (thymine-thymine dimer)
- Opening of DNA double helix the XP-C/23B protein complex recruits transcription factor TFIIH the ATP-powered helixase starts to paritally unwind the double helix and then XP-G and RPA protien bind and further unwinf and stabilize the helix forming a buble
- Excision of DNA lesion segment; endonuclease XP-F and XP-G cut on the damaged strand at points 24-32 bases apart on each side the endonuclease then makes two DNA cuts which releases the DNA fragment with the damaged bases damaged DNA fragment is degraded to mono nucleotides
- Gap repair filled by DNA polymerase then sealed by DNA ligase
Are exonuclease required to make the DNA gap
NO
What is transcpriton couple NER
The mechanism operates slightly different in the regions of genome being activly transcrpited
What is different about transcription-coupled NER and global
In recognition of DNA lesions they use RNA polymerase as a sensor then recurits CSB which then triggers the opening of the DNA helix and recruits RFIIH and other factors (step 2 and 4) ( doesn’t use XP-C/23B complex)
What does Xeroderma pigemtosum name
Many proteins involved in NER mechanisms
What disease does XP protiens cause
Rare autsomal recessive disorder of DNA reapiar that is characaterzied by sun sensitivity and UV radiation-induced skin and mucous membrane cancers 1000 more likely to develop skin cancer when exposed to the UV rays in sunlight mutated XP genes and NER cannot repair DNA damage induced by UV radiation
What can gamma and X-rays do
Much more energetic than UV light and can ionize the molecules around DNA especially water and form highly reactive free radicals
What can these radicals do that are caused by ionizing radiation
Attack DNA by altering bases and breaking strands
How can single strands be repaired
BER
MER
NER
What are the two ways to deal with dsDNA breaks
NHEJ
HR
What does NHEJ involve
Direct ligation of two DSB little or no sequence homology required
What is a key factor of NHEJ
Doesnt need a sister chromtid so it can occur during any phase of they cycle
What are some downsides of NHEJ
Limited loss of genetic information resulting is short deletions ligation of the incorrect ends if multiple DSB are present generate large deletions or chromsomal rerrangments
What does HR need
Template for repair which allows it to restore any missing genetic information largely accurate
What is used at the repair remplate in HR
The sister chromatid
What cell cylce can HR occur at
S G2
What are the steps of NHEJ
- DNA end binding and bridging a complex of KU proteins and DNA dependent protein kinase binds to the ends of DSB KU hetrodimer forms a ring and enricles the duplex DNA that forms a brige or synapse between the broken end
- Terminal end processing KU hetrodimer and DNA-PK recruit and activate other proteins that are required for the processing of DNA ends including exonuclease that trim overhanging ends
- Ligation the blunt DNA ends are joined together by DNA ligase
What is the KU heterodimer
KU70 and KU80
What is the steps of HR
- DSB recognition: DSB sensors are poly(ADP) polymerase PARP and MRN complex which also mediate initial DSB resection
2.Extensive DNA resection: this occurs with the help of several protiens including BRCA1 activation of all protiens is controlled by ATM (required to form overhanging 3’ ssDNA ends coated with RPA) - Assembly of RAD51 filaments the overhanging 3’ ssDNA needs to be activated RPA displacement with RAD51 which is coordinated by BRCA2
- DNA invasion: RAD51 catalyzes invasion of 3’ ssDNA ends into homologous DNA of the homologues chromsome
- Hoilday junction formation and resolution Holiday junctions are branched DNA structure formed by strand exchange during recombination that provides DNA templates to repair DNA lesions. The repair strands seperate
What is a sensor of DSB in NHEJ
KU hetro dimer
What defects in DNA reapir by HR caused by
Inheritance of one mutant allele of the BRCA1 and BCRA2 genes results in predisposition to breast and ovarian cancer
What does BRCA1 controal
Step of extensive DSB resection
What does BRCA2 control
assembly of RAD51 filaments and invasion
