DNA repair

Question 1

what is hNPCC testing?

Answer 1

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

Question 2

what can modify bases?

Answer 2

alkylating agents can methylate bases; deamination removes an NH2 and replaces it with oxygen; oxidation replaces Hs with Os or OHs

Question 3

what are the alkylating agents?

Answer 3

methyl and ethyl methane sulfonate–> MMS and EMS common mutagens in the lab

Question 4

what are some deaminating agents?

Answer 4

peroxides, nitrous acid, formaldehyde

Question 5

what are some oxidizing agents?

Answer 5

peroxides, superoxides, and other rare chemicals

Question 6

cytosine deamination pathway

Answer 6

C:G–>U:G–>U:A–>T:A

Question 7

adenine deamination followed by enzymatic xanthine oxydation

Answer 7

A:T–>HX:T–>X:T–>HX:C–>G:C

Question 8

guanine deamination pathway

Answer 8

G:C–>X:C–>G:C (no mutation)

Question 9

5-Me cytosine deamination pathway

Answer 9

5-Me-C:G–>T:G–>T:A

5-Me-C accounts for 5% of all cytosines in a cell

Question 10

waht is the purpose of human glycosylases?

Answer 10

each one is highly specific to bind and remove a subset of modified bases

Question 11

what are the TLS pols in bacteria?

Answer 11

Pol IV and V

Question 12

10 different types of TLS pols in humans

Answer 12

ya

Question 13

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

Answer 13

ya

Question 14

what are the humans TLS pols?

Answer 14

Xi, Eta, Iota, Kappa, Zeta, and Rev1

Question 15

how does Xi function during TLS?

Answer 15

incorprates bases randomly

Question 16

how does Eta function during TLS?

Answer 16

put two A’s across from an unfixed thymine dimer

Question 17

how does Iota function during TLS?

Answer 17

synths. past 6-4PPs and abasic sites–>checks pairing along side face of major groove (Hoogsteen DNA)

Question 18

how does Kappa function during TLS?

Answer 18

synths past G-G interstrand crosslinks (alkylating agents);

Question 19

how does Zeta function during TLS?

Answer 19

extend synthesis from mismatched bases

Question 20

how does Rev1 function during TLS?

Answer 20

NOT a DNA pol, rather a deoxycytidyl transferase–>incorporates C and C only, no matter the template

Question 21

can possibly inhibit TLS pols for cancer treatment–> cells too damaged and no cellular desperation mech to get past the damage–>no proliferation

Answer 21

ya

Question 22

what are the proteins involved in prok MMR?

Answer 22

MutS, L, and H

Question 23

waht is MutS’s role in prok MMR?

Answer 23

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

Question 24

what is the purpose of MutL?

Answer 24

dimerizes and is recruited by MutS; recruits MutH

Question 25

what is mutH?

Answer 25

endonuc which can only cut at unmethylated GATC seqs–>so can only cut the new strand

Question 26

what is the purpose of Dam?

Answer 26

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)

Question 27

what is UvrD’s role?

Answer 27

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

Question 28

what is photolyase’s role?

Answer 28

breaks apart thymine dimers; repair is called photoreactivation as they use blue light to break about the dimer

Question 29

Describe NER

Answer 29

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

Question 30

human NER steps

Answer 30

step 1. damage recognition; 2. local unwinding; 3. strand dual-inclusion 4. repair synth and strand ligation 5. repaired DNA

Question 31

step 1 of NER in humans

Answer 31

for GG-NER: HHR23B and XPC bind to bulge; for TC-NER, RNA pol II and CSB bind bulge

Question 32

step 2 of NER in humans

Answer 32

local unwinding: TFIIH, XPB, XPC, XPA, and RPA unwind DNA

Question 33

step 3 of NER in humans

Answer 33

strand dual inclusion: ERCC1/XPF and XPG cut the sites by XPB and XPD

Question 34

step 3 of NER in humans

Answer 34

repar synth: RFC, RPA, PCNA, Pol delta/epsilon, and ligase create reapired DNA

Question 35

briefly describe xeroderma pigmentosum

Answer 35

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;

Question 36

briefly describe cockayne’s syndrome

Answer 36

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

Question 37

treatment for XP (and CS and sunburn)

Answer 37

–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

Question 38

describe BER

Answer 38

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