DNA repair Flashcards
what is hNPCC testing?
hereditary nonpolyposis colon cancer;
indirect test 1: microsatellite instability– 5 microsats are tested, if 2 or more are found to be variable, it’s likely the individual has HNPCC
indirect test 2, immunohistochemistry (Abs): tumors are tested for the presence of MSH2, MLH1, PMS2 (mutL hom), and MH6–>Abs not binding means they aren’t expressed or epitope is mutated
what can modify bases?
alkylating agents can methylate bases; deamination removes an NH2 and replaces it with oxygen; oxidation replaces Hs with Os or OHs
what are the alkylating agents?
methyl and ethyl methane sulfonate–> MMS and EMS common mutagens in the lab
what are some deaminating agents?
peroxides, nitrous acid, formaldehyde
what are some oxidizing agents?
peroxides, superoxides, and other rare chemicals
cytosine deamination pathway
C:G–>U:G–>U:A–>T:A
adenine deamination followed by enzymatic xanthine oxydation
A:T–>HX:T–>X:T–>HX:C–>G:C
guanine deamination pathway
G:C–>X:C–>G:C (no mutation)
5-Me cytosine deamination pathway
5-Me-C:G–>T:G–>T:A
5-Me-C accounts for 5% of all cytosines in a cell
waht is the purpose of human glycosylases?
each one is highly specific to bind and remove a subset of modified bases
what are the TLS pols in bacteria?
Pol IV and V
10 different types of TLS pols in humans
ya
TLS pols are usually only transcribed when the cell senses damage–> MUST be tightly controlled, do not want loose/error protein pols flooding the DNA replication system; specialized for certain types of damage
ya
what are the humans TLS pols?
Xi, Eta, Iota, Kappa, Zeta, and Rev1
how does Xi function during TLS?
incorprates bases randomly
how does Eta function during TLS?
put two A’s across from an unfixed thymine dimer
how does Iota function during TLS?
synths. past 6-4PPs and abasic sites–>checks pairing along side face of major groove (Hoogsteen DNA)
how does Kappa function during TLS?
synths past G-G interstrand crosslinks (alkylating agents);
how does Zeta function during TLS?
extend synthesis from mismatched bases
how does Rev1 function during TLS?
NOT a DNA pol, rather a deoxycytidyl transferase–>incorporates C and C only, no matter the template
can possibly inhibit TLS pols for cancer treatment–> cells too damaged and no cellular desperation mech to get past the damage–>no proliferation
ya
what are the proteins involved in prok MMR?
MutS, L, and H
waht is MutS’s role in prok MMR?
ATPase which dimerizes and bind to DNA backbone to scan for mismatches; a change in DNA conf leads to a change in prot conf leads to enhanced ATP binding–>slower ATP hydrolysis–>stalls and waits for MutL
what is the purpose of MutL?
dimerizes and is recruited by MutS; recruits MutH
what is mutH?
endonuc which can only cut at unmethylated GATC seqs–>so can only cut the new strand
what is the purpose of Dam?
DNA adenine methylase; methylates the A in the GATC, but only ~10 minutes after replication has finished (gives enough time for MutH to do its job)
what is UvrD’s role?
weakly processive helicase–> 3’-5’ helicase activity; initiates DNA unwinding at nick produced by MutH and moves towards the MutS/L complex; exonuc removes the strip of ssDNA and replication machinery fills in the gap
what is photolyase’s role?
breaks apart thymine dimers; repair is called photoreactivation as they use blue light to break about the dimer
Describe NER
nt excision repair; UvrA recognizes lesion as a dimer; UvrB flexes DNA creating a bubble at the lesion to recruit UvrC, UvrC cuts 8 bps upstream adn 4 bp downstream of lesion; UvrD is a helicase which removes the DNA between the cuts; pol and ligase fill in
human NER steps
step 1. damage recognition; 2. local unwinding; 3. strand dual-inclusion 4. repair synth and strand ligation 5. repaired DNA
step 1 of NER in humans
for GG-NER: HHR23B and XPC bind to bulge; for TC-NER, RNA pol II and CSB bind bulge
step 2 of NER in humans
local unwinding: TFIIH, XPB, XPC, XPA, and RPA unwind DNA
step 3 of NER in humans
strand dual inclusion: ERCC1/XPF and XPG cut the sites by XPB and XPD
step 3 of NER in humans
repar synth: RFC, RPA, PCNA, Pol delta/epsilon, and ligase create reapired DNA
briefly describe xeroderma pigmentosum
disease where individals have no ability to repair damage to DNA from UV light; rate of skin cancer is much, much higher; of the eight XP gene/proteins, 7 of them have mutations which affect NER;
briefly describe cockayne’s syndrome
not associated with cancer; dwarfism, deafness, microcephaly, mental defects, osteoporsis, sun sensitivity, degenerative blindness and cataracts= aging?; no reason yet as to why CS might come from issue with transcription-coupled repair
treatment for XP (and CS and sunburn)
–minimize UV damage; insert photolyase specific for cyclobutane dimers from cyanobacteria, overexpress in e coli, package into liposomes–> put on skin–helped reduce cancerous lesions
describe BER
used for repairing damaged DNA that do not distort DNA structure–>often detects by sensing in minor groove; nonstandard base removed by specific DNA glycosylase; AP endonuclease cuts 5’ to missing base; dRpase (deoxyribose-5-phosphatase) removes sugar/phosphate; pol beta fills in and ligase seals
