Lecture #4 - DNA mutation, Damage, and Repair

Question 1

DNA mutation and Repair

Answer 1

Mutation and damage are consstattly occuring

Repair mechanisms have evoloved to maintain genome integrity

Question 2

Why study mutation

Answer 2

1. No mutation = no evolution
2. Somatic Evolution (Ex. mutation that causes cancer –> chnages in the geome caises cells to dvivde uncontrollably)
3. Genome has a lot of repair proteins (The genome is tellus to to study DNA repair based on the amount of work the genome puts into repair means that it is important)

Question 3

DNA mutation + Damage + Repair Summary

Answer 3

DNA repair is essential - the integrity of the genome is crtical for the function of each cell and its progeny

DNA replication is inheritley imperfect EVEN with DNA polymerase proofreaidng ability

DNA is subject to damage from internal and external sources

DNA repair systems probably appears shortly after DNA apeared

DNA repair systems are diverse and efefctive
- ONLY 1/1,000 incidents of DNA damage results in mutations
- Cells devote many genes tp DNA repair (Ex. E.coli has 4,400 genes and 100 code for DNA repair proteins)
- Repair often enlists replication and recombination functions
- Failure to repair DNA damage leads to genome instability and disease (Especially cancer)

Question 4

Intirinic things that affect mutations

Answer 4

ALL independent of external factors
- Extrenal would have been chemicals + radition BUT these are ALL intrinsic

1. Tautomerization
2. Depurination
3. Hydrolytic Demanination

Question 5

Tautomerization

Answer 5

Tautomer formation can lead to mis-incorproation of nucleotdes

IMage:
A - normal A-T base pair (Most energetically favoriable configuration)
B - Tautomer - forms Imino –> gain H on the N in the middle of teh ring –> Double bond flips around (Forms a double bond C-NH AND NH loses an NH becaue it was NH2)

Change causes a reversal of teh H-Bond donar ad acceptor characteristics
- IN tautmer the top N is the acceptor and te bottom is the donor –> ALSO the imino group to pair with C

Get C-A mismatch –> A-C mismatch can be recognized by DNA repair enzymes (IF not repaired then when replication C-A because C-G in the new stand)
- Doesn’t often cause too mamy probelms bevause have enzymes that look for mismatchs

Question 6

Frequenceu of Tautomers

Answer 6

Have less than 0.01% of bases in the minor tautomer form BUT in the human genome there are hundred throusand bases in thsi form at any momment
- Have a low percent of tautoemrs BUT can still have a high amount in numbers because have so many dNTPs in cell

Question 7

Depurination

Answer 7

Common in G/C NOT common in A/T

G has linage to phosphae backbone BUT this link is not 100% stable so it can be hydrolyzed (carbon is atcked by water)–> get depurinated sugar –> upon replication you have a random base substituton or base skipping opposite the missing base –> END have a substitutioon or delation
- NOT stable BUT have many based aeround where the G base was dropped and have the depurinated sugar

THIS is the most frequent spontenous alterations (10^4 per cell per day)

Question 8

Hydrolitic demaamination

Answer 8

Hydrolytic deamination results in nucleotode mispairing
- Have Hydrolytic demaination of a cytosine to get a uracil

Have a cytosin whee the NH2 is attacked by water –> knock out the NH3 + forms a carbnyl where the NH2 was –> Forms a uracil
- NOW have a U in DNA –> f that replicates the U is paired with an A (instead of having a C that would pair to a G) –> On teh second round of replication teh A pairs with a T –> Get CG to AT

Fidelity of DNA polymeras wants to catch this before the next round of replication

Question 9

Envirnmnetal Damage

Answer 9

1. UV light
2. Intercalating Agents
3. Envirnmental and endogenous alkyl donors

Question 10

UV light

Answer 10

UV can make 2 types of adducts:
1. Cyclobutane dimer – when have 2 pyrimidines next to each other on the same strand –> Adjacent T BPs become joined by a 4 memebered ring (4 memebered ring is not stable BUT because the bases are close it can still happen)
2. 6-4 photoproduct - form a single bond

BOTH cause pyrimidine dimers which perturb the base stacking interactions between DNA bases –> Distroting the phosphodirester backbone and preventing DNA replication

Formation of ither adduct is bad for the double helix
- Formation of both structires peaks at 260 nm BUT you can still get them at 300 nm

Question 11

Intercalating agents

Answer 11

Intecalating agents = planer conjugating ring system hat can interact with DNA by interacalting between adjacent BP

Planer surface of BP and hydrophobic molecule (Intercalating agent) can escape aqeous envirnments = the interacalting agent goe sbetween the bases
- Example - Ethidium bromide goes between DNA bases = moves the bases farther apart

When the bases are pushed apart and partly unwind the DNA helix due to agents –> DNA replication can make a mistake by thinkning that it is the right spacing –> Causes addition or subtraction of BP
- Add or subtract depending on if template or new strand
- Get single nucleotides insertion or deletion

Question 12

Why does ethidium bromide work

Answer 12

Ethidium bromide can work because UV is absorbed by the bases –> UV energy is trasmitted in non-radiated reaction to ethidium with floruenscene

Overall - non-radiative trasnfer of energy

Question 13

Envirnmental and endogenous alkyl donors

Answer 13

Envirnmental and endogenous alykl donors result in nucleotode mispairs

Alkylation (Ex. CH3) can be added to the 6th position of Guanine

Image - shows alykaltion (Add CH3) –> alylated G is more likley to pair with T (NOT a C) –> during the second round of DNA replication the T pairs with an A

END have GC –> AT

Question 14

Use of alkyl donors in labs

Answer 14

IN LABS allkyl donaors are used for mutatinos screen -

Ex. EMS is a commonly used muatten in foward genetoc screens
- EMS ethyl group is transfered –> causes ethylated guannine

Question 15

What system is used for studying mutagensis

Answer 15

Best system to study mutagensis is Lac repressor –> Determines the types of mutations caused by exposure to particular chemicals or radition

Lac Operon - mediates the utilization of lactose as carbon
- Codes of beta-galatosidase –> Cleaves lactose
- Codes for genes that digest lactose
- Lac operon genes are ALL under the Lac I Repressor (Upstream of the promoter) –> Lac I codes for the lac repressor –> Lac reprsor binds to the operater (Lac O) –> Repressor binding to Lac O inhibits transcription
- Lac represor make sure that you are not making gene product when there is no lactose around
- IF have lactose –> lactose binds to the repressore –> repressor can’t bind to DNA –> have transcription to get the gene product

Question 16

Detecting mutations in Lac repressor model

Answer 16

X-Gal is colorless –> mix X-gal into agar of petri plate

X-gal is cleaved by beta-galatosidase –> product of cleave is a blue compund that accumilates in colony
- On plate can see blue and white colonies
- White colonies are NOT making B-galactosidase because they are not clecaing to form a blue compund –> means the white colonies have intact represore because not mkaing lac repressor
- Blue colonies - have mutatnt repressore = can’t reprressore = get Lac operon expresssion = get beta-galatosicase = get blue compound due to cleavage by beta-ga;atosidase
- When have no lactorse on the plate the lac operon should be repressed

Overall - can see what has the lac repressor and what doesn’t

Question 17

Spontenous mutations

Answer 17

Spontenous mutations are Non-random (Mutation occirs in a distict pattern in both spintenous and induced mutations)

Image - shows the lac I gene
- Each square or circle have a muations –> see pile of red squares/piles of blue squares (All red mutations are idetocal BUT indepent ; same with blue)
- See series of deletions in non-random way

Question 18

Most common location for mutations

Answer 18

Most common location for mutations are small regiosn with repetative sequences

Example - CTGG)
- Most common mutation = amplification –> because CTGG is not 3 nuceltodes you get framshift
- Second most common = loss of 1 copy –> also cuases frameshift

Mutation occurs because of slipage on new or template strand

Question 19

Most common mutation in humans

Answer 19

Most common muttaion in humans = slipage of repetative sequences

Question 20

Most common deletion

Answer 20

Most common deletion = have repeats of teh same sequence at terminal

Image - GTGGTGAA repeat in blue box
- Hae a recombination event of DNA slipage –> results in deletion that leads to one copy of repat with nothing in between

Most common structre of CNVs:
1. Have repeated sequeces where the region between is removed and there is only 1 repeat left
2. The whole thing gets copied and have two idetical sequences (Repeat –> Uniuq DNA –> repeat –> Unique DNA –> Repeat DNA)

Question 21

DNA mutation themes

Answer 21

DNA mutation themes tend to span across different organisms

Mechanism of repair is usually conserved

Question 22

Are point mutation random

Answer 22

Spontenous point mutations are NOT random

Chart - shwos the most common poiint mutation –> See that point mutations are more common in places where there is a methylated C

C can be demainated –> because uracil -> Uracil is recognized by Uricil N-glycoidase –> Uracil N-glycosidase sees U in teh DNA and cuts out the deoxy base and the cut is recognized by other enzymes

Mythlated C (made after DNA is replicated) can be deaminated -> get T
- THIS is dangerou sbecause Y is not indistgushable from other T = can’t recognzie it and cut it out
- ONly way to recgiize is by e TG mismatch which would form BUT the enzymes need to know if teh T or teh G is correct
- ALSO don’t always get correction done in time or correctly –> cases mtehylateC to cause GC–> AT mutations

MOST commmon point muttaion in humans

Question 23

Studying damage and mutatgeniss in humans

Answer 23

Can still use Lac system – LacI gene can be shuttled beteween bateria and mammaliln cells through plasmids to deterine the spectrum of mutation in mammalian cells

Use the Lac system –> Put DNA into mammalian cells –> do mutogensis –> allow repair to happenin mammalian cells –> Put DNA into E.coli –> Do analysis

Take HEK293 cells –> transfect with plasmid carying the Lac I gene (NOW have plasmid that encodes the lac repressor) –> treat with EMS –>recover the plasmid –> Put the plasmid back in E.coli for functional and sequence anylysis–> Look for plasmid where the Lac repressor is mutated
- E.coli used have no repressor gene = need repressor from the plasmid (Plasmid added t the E.coli will be mutatnt or funcctioning depends on if it was fixed by DNA repair mechanisms when in mammalian cells)
- IF the plasmid is non-function wit will trurn X-gal blue = can pick colny and sequence (non-fucntioning blue?)

Question 24

Result of putting lac repressor in mammalian cells

Answer 24

When done - See ALL the mutations ae GC–> AT - Means the mutagen has specifcty in temrs of what changes it produces (results aligns with EMS as alkylting agent)

Question 25

Ames test

Answer 25

Quantofys reversion from His- to His+ of salmonella strains with defined muttaions Tests for the mutageic potencey of a treatment (used to monitor envinrmntal samples) Take rat liver extract -> put with salmenella srain that requires Histadine --> Mix each salmonealla strai with liver extract and mutagen --> put teh strains on plates that lack Histadine --> IF the mutagen can convert the mutation (C --> T in mutatnt but mutagen would mke the C back to T then it would rmeove the oxitrophy marker to WT that cna make histadine --> Colony will grow even though plate has no histadine --> Count the colonies and see potencey of mutations - Salmonella used can't make histadine (they are histadine oxotrophes --> require Histadine to be added to the agar plate - Know the sequences that cause the oxotrophy in salmonella

Question 26

Why use liver extract in Ames test

Answer 26

Because the liver is a detoxifying organ - Uses CYT-p45 enzumes to detoxify anything that is ingested Put mutagen with liver extract = gives the mutagen an oppertunity to be trasnformed to something that is more similar to what i would be in living organism - Liver can detoxigy BUT can also modify the mutagen o make it more mutagenic

Question 27

Aflotoxin

Answer 27

Aflotoxin = potent mutagen/carcinigen produced by fungi + molds - Grows on corn + peanuts + other plants - Aflotoxin can be detected by the Ames test When activated by cyt-p50 in liver alfotoxin gets worse --> source of HCC

Question 28

How can DNA damage occur?

Answer 28

DNA damage can occur through: 1. Chemical reactions inherint to DNA striucture (Ex. Tautomerization + Depurintaion + Deamintion) 2. Chemical reactions induced by metabolic products (EX. Alykalting agents + Oxidizing agents) 3. Envirnmental factors (Ex. ionizing radtion + Genotoxic materials)

Question 29

Answer 29

A is not right because can have damage that is correct = no mutation DNA damage rarley leads to mutaion

Question 30

DNA repair by direct reversal

Answer 30

1. Photolysases 2. Ada enzymes for alkylatiion

Question 31

DNA repair (Direct reversal)

Answer 31

Repair by direct reversal of DNA damager - Photolysases retrun UV-induced dimers to their original monomeric state - DNA base products of interaction with ROS and free radicals Have DNA photolyses (type of flavoenzyme) --> captire blue light (lower energy than UV light) to reverse lsions - NOT in placental mammals (not in humans) Have UV radiation product (Ex. cyclobutane ring) --> can be reversed by photoenzymes - Enzymes use light harvetsing as a co-factor that binds to adducts in DNA and uses visible light energy to reverse the reaction

Question 32

Repaur by direct reverasal (Alyklation)

Answer 32

Image - Damage is teh dimanonds Can revserse damage done by alkylation - E.coli gene Ada (Adpatove response to alylation) encides O-6-methylguanine DNA methyltransferase (Ada Protein) - Have a suicide protein (methyltransferase) that recgnizes the alykated base --> protein attacks the alykl grooup --> protein transfers the alkyl group to a cysteine in active site on itself (NOW have alkyl on protein not on DNA) Methyltrasferase protein controls its own transcription -> alkylation of protein (occurs when have a lot of alykation damage in cell) = activates transcription to make more of the protein (Make more protein when the cell needs more of the protein) - Methylated Ada activates transcrtion of Ada gene and a glycosylase gene for methylated bases

Question 33

Repair stragedies involoving DNA syntehsis or recombination

Answer 33

Ways to fix DNA: 1. BER - gets rid of damaged base ith N-glycosylase - N-glycosylase cuts the base off the sugar --> Site with no base is recognized by other enzymes that cut the DNA --> DNA is fixed by poymerase 2. Oligion NER (Excises 1-20 BP) -- IF have damage on one strand = make incision upstream and downstream of damage --> remove oligionucleotides --> Single strand is resynthesized by Polymerase and sealed by ligase 3. Tranlesion repair - Used during DNA replication - DNA plymerase is bad at dealing with lesions/modifcations on DA --> IF have a lesion during DNA replication --> DNA polmerase will stop BUT the cell doesn't like this and wants tp keep replicating = cell using a bypass polymerase that is error prone --> Bypass polymerase sythesizes across the modified DNA --> Bypass polymerase can introduce incorrect bases --> ONce bypass polymerase gets across modification the replication polymerase will come back - Last resort 4. Recombination - Use infomration from duplicated double helix (Ex. seperate chromoeome_ --> chromsome copy can be donated from a good copy to fix the bad copy

Question 34

Base Excision Repair

Answer 34

Overall - Have a damaged base --> N-glycosylase remives base --> removed base is recognzied by AP endonuclease --> Encoducleases cut --> DNA polymerase fixes 1. Recognition of damaged base by a DNA glycosylase 2. Removal of damaged base by DNA glycosylase 3. Recognition of the abasic site by the AP endonuclease 4. Cleavage of the phsophodieter bonds blanking teh abasic site 5. Replacment of excised nucleotide by DNA polymerase and ligase

Question 35

Glycosylase recogniztion in BER

Answer 35

Glycosylases can swivel the base out of the helix --> look at base --> snip off base - Base flipping by glycosylase exposes the damaged base THN can cleave the glycosidc bond

Question 36

Nucleotide excision repair

Answer 36

Oligionucleotide excision repair acts on bulky lesions that distort the DNA helix - Can repair thymidine dimers (redundent repair) Start - have bulky lesion 1. Recognition of lesion by UvrAB comlex - E.coli UvrAB recgine lesions by assing the bendability of DNA (see distrotion in helix) 2. Opening of DNA around damaged site by UvrB helicase and release of UVrA 3. Recruitment of UvrC and cleavage by UvrC 3' and 5' damage site - UvrC comes and makes nick on either side 4. UvrD removes damaged DNA and UvrC is released 5. Resythesis of excised DNA by DNA polymerase and ligation (green in image)

Question 37

Defects in NER

Answer 37

Defects in NER by mutating proteins in NER associated with disease in humans - ALL the disorders have high frequncey of skin cancer because hvae UV mutations that are not being repaired (Ex. Trichytdacsty and Xeroderma pigmentosa In humans - NER pathway has more proteins doing fewer things - Overall - Snip DNA and resythesize

Question 38

Post replication mismatch repair

Answer 38

Post replication mismatch reapir exists in gram negative bacteria (Ex. E.coli) Question - if a mistake occurs (DNA polymerases makes a mistake or tautermization occurs) can you go back and fix it? - Answer - YES because E.Coli DNA polymerases makes 1 error per 10^7 BP BUT has a mutation rate of 1 per 10^9 bases --> MEANS 99 out of 10 mistakes are fixed HOW are misatkes fixed - done by post-replicative system - Post-rpelicative system also exists in humans + plants + fungi - Overall - system looks at new DNA to see if it is ok

Question 39

How does bacteria know newly synthesized DNA vs. Template DNA

Answer 39

In bacteria - post replicative mismatch repair mechansims distiguish template DNA from newly syntehsized DNA based on under-methylaton of newly syntehsized DNA - Driven by meythylation - dNTPs do not have meythl on them = all NEW DNA is unmethylated Image - OLD DNA is methylated at A (seen GATC where both A have methyl) --> when DNA is replicated the p[roduct strand won't have a methyl - DAM methylation meythylaes the newly syntehszied strand by finding the methylated sites --> puts methyln on A in new strand - BEFORE the DAM adds methyel - have a faik safe mechnaism that can look for mismatch + use the fact that the old srand is methylated to know which strand is the misatje and which strand has correct base = corrects old strand

Question 40

Paul Mdrich

Answer 40

Biochemist who elucidated the mechanism of post-replication mismatch repair

Question 41

Post-replication mismatch repair in humans

Answer 41

ISSUE - IF have a GT mismatch NOT nearby a methyalted A (meythlayed A is 100 BP away) --> how does the enzyme know the correct old and new strand if the mark for the old vs. new strand is far away Answer - have a series of sliding clamps around DNA (Clamp is on the DNA in asymetruc way) --> Can know what is old and new up to the point the mutation is idetofied - Clamp slides on the DNA in a way that preserves the infomration of which strand is old vs. new (Clamp is asymetric at place of asymetric methylation) - Enzymes that do the repairing can engae the ring in asymetic way = enzymes know which way to apparch the DNA based on how the ring is sitting on the DNA - The clamp ring maintains orinetation with respect to old and new strand even though it slides

Question 42

Associated of mutation in MMR genes

Answer 42

Mutation in mismatch repair (MMR) genes are associated with heridary non-polyposis in colorectal cancer

Question 43

Trans-lesion DNA synthesis

Answer 43

Trans-lesion DNA syntehsis = mechasnim for bypassing a damaged base wothout correcting it LAST resort - IF have a DNA modification that blocks DNA polymerase --> Trans-lesion polymerase comes in for THAT interval to syntehsize acoss modificed place --> THEN hand back to DNA polymerase - Trans-lesion polymerase is highly mutagenic

Question 44

Issue with Trans-lesion DNA syntehsis

Answer 44

Trans-lesion DNA syntehsies is done by an error proone DNA polymerase Trans-lesion Polyemrase permits synetshis across unrepaired DNA lesions - Carried out by an error prone DNA polymerase - Error rates are 10^-3 vs. replicative DNA polymerase error rate is 10^-7 (error rate is 1 in 1000 --> have high mutation rate by having error prone polymerase take over for a breif time) - Error prone DNA polymerases are tyoically induced by DNA damage

Question 45

Interstrand DNA cross links

Answer 45

Interstand DNA cross links = type of damage that is repaired by Fanconi Anemia proteins Have repair machine in humans and bacteria - humans have modifications that cross links teh two DNA strands (VERY bad for replication because the replication fork can't pull strands apart because they are now linked by a covalent bond) - Uses many proteins that recognizes the strands joining togetehr and will break the strands apart - Associated with Fanconi Anemica --> have cross links on all of the cells = get multi-organ dysfunction

Question 46

Repair Based on DNA base perubuations Summary

Answer 46

1. Mismatches or bulky adducts are reocognzued by porteins that sense chnages in DNA structure pr flexibility 2. Specific pathways have evoloved to repair different types of damage 3. In the absense of repair - an error prone DNA polymerase replicates across damaged bases

Question 47

What causes Double strand breaks

Answer 47

1. Ioniing radiation (High energy radiation) 2. Replication fork collapse

Question 48

How to you repair dsDNA breaks

Answer 48

BEFORE - all methods fixed single strand issues To fix double strand: 1. Non-homologeous end joining (NHEJ) - Puts fragmenst togetehr because have two seperated ends (seperated ends are bad - Ex. Can't do DNA replication) - Inheritley error prone (often lose bases) - Done using CRIPSR editting -- get dsDNA break that is repaired in error prone fashion - Used in antibodu and TCR gene rearengment 2. Homology directed repair - Homologous recombination

Question 49

Homology directed repair (Overall)

Answer 49

Use other chromosme OR in bacteria can use partially replcated DNA uses the newly sntehsized DNA to correct inforation Uses correcct infomration to put the peice togetehr correctley - Get precise joining using infomration from other homology

Question 50

Issue with NHEJ

Answer 50

NHEJ is inheritle a mutogenic process (sloppy system) Two ways this can happen: 1. Cut and nucleotodes wil degrade form the end --> peices are then ligated back together - Often lose 205 BP 2. Degredation of 1 of the 2 strands (using single stranded nucleases) --> IF overhang has homology they can pair --> single strands get cut away --> ligate the peices togetehr - Example in image - Homology (TGAC on upper strand can bind with ACTG) - STILL get deletion - Microhomology mediated end joining?

Question 51

Homology directed repair (HIS NOTES)

Answer 51

Overall - Resection of >50 nt --> Synapsis --> Syntehsis dependent strand annealing Steps: 1. Generation of ssDNA (resection) at the site of dsDNA break 2. Pairing of one single strand end with teh intact duplex to form heteroduplex 3. Repair of damaged duplex by DNA sythesis using the undamged temaplte 4. Seperation of the two duplexes

Question 52

Homology directed repair (MY NOTES)

Answer 52

Overall - uses another chromosome to donate infomration - Only way to donate infomration is by Watson/Crick BP - Can correct mismatch repair - NOTHING is lost - IMAGE - green was donated from the red good copy How do you get information to the chromsome that needs: 1. Resect strands = get ssDNA 2. Strands invade the double helix 3. Want syntheszie form the 3' end to extend the region further = new BP can be broken --> new ssDNA can g back to orginal chrosome and now has the region that includes the break so wen BP on the distake side of the brak with ssDNA it can close the gap with all of the infomration 4. Gaps sealed by DNA polymerase and ligase HOW does the strand find teh correct sequence if it is a small part in a vast genome --> Uses an enzyme that can catykyze strand invasion - Enzyme pulls strands apart and allows ssDNA to sniff around to find the correct BP --> When finds the right BP it forms semi-enzymes that catylyze strand invasion - Similar to system used in CRIPR to seach in genome for correct place

Question 53

What is used during single strand excange

Answer 53

Single strand exchnage is mediated by specilaized reocmbinases (Strand exchange proteins Image - shows strand exhange proteins (RecA proteins, RAd51 in Euk, DMc in meiosis) - Starnd exchange proteins binds to ssDNA and catykyze invasion of the double helix by ssDNA - RecA + ssDNA complex nucleofiliment binds to a dsDNA which extends the duplex --> displace one strand of the duplex by invasing ssDNA and D loop is formed - Need single strand exhnage enzymes + ATP for strand exchange

Question 54

BRACA1

Answer 54

BRACA 1(tumor supressor gene) = breast cancer suseptability protein - Normally BRACA1 = facilitates 3' end resction - Mutation increases risk of breast and ovarian cancer In S phase - BRAC binds to dsDNA break sites -> BRAcA1 modifies histons --> Modifcations leads to resuritment of chromatin remodelers + resection enzymes + RAD51 - Promotes homology-directed DNA repair and inhibits NHEJ - After resection RAD51 mediates invasion BUT in order for this to happen you need BRACA to recognize the dsDNA break

Question 55

Pathways to repair dsDNa breaks Summary

Answer 55

1. NHEJ is an error prone pathway that does not require extnsive homology 2. Homology directed repair requires a sister chromatid or homologous chromsome 3. Strand-exchnage recombinases assmeble on single strands to probe dsDNA for regions of homology 4. The cesision to use NHEJ or HDR is regulated by sensors that nitially recognize dsDNA breaks

Question 56

INducible DNA damage response

Answer 56

INducible DNA damage response = SOS Sensing damage is important because repair enzymes can be recuited if needed Normally - there is a basal level of repair proteins BUT if there is a lot of damage you want more enzymes - Synthesis of repair enzyms is regulated in this way

Question 57

SOS system

Answer 57

In E.coli RecA protein mediates strand invasion in HDR BUT also senses DNA damage Normal cell - there is no ssDNA (nly have transient ssDNA during replication) - Normally LexA binds to promoter region of DNA repair genes = keeps expression of DNA repair genes low In DNA damage - replication forks stalls (leaidng strand might continue but the lagging strand gets blocked) --> GEnerate lots of ssDNA After generation of ssDNA --> RecA finds substrate ssDNA and binds to the substrate ssDNA --> Binding causing conformation change --> conformation chnage reveals a protease site --> protease site it reveals can cleave LexA repressor (RecA cleaves between the two domains in LexA) --> Clwaved LexA can't bind to the promoter of DNA repair genes --> Get expression of teh DNA repair genes --> Get formation of repair proteins (Ex. Get UVRA, UVRB, UVRD)

Question 58

How does SOS system turn off

Answer 58

When DNA is repaired and cell returns to irginal state there is little ssDNA --> RecA protease cativity stops --> get increase in LexA repressor --> repress expression of DNA repair genes

Question 59

Mechanism of action of RecA in SOS

Answer 59

1. Cleaves LexA 2. Cleave lysogenic bateriaphage repressors (Ex. cleaves lamda repressor) --> leads to iniduction of lysogen --> Allsows the progeney of the bacteriaphage to escpae from the damaging envirnment of the bacterial host - Can get excision of the integrated becateriaphage genome and activation of the lytic pathway

Question 60

How fast does SOS pathway occur

Answer 60

Chart - shows induction of SfiA following UV radiation of E.coli - SifA = cell cycle arrest gene (stops the cell cycle and fix DNA to stop replicaton = induced during DNA repair ; slows dow cell cycle during DNA repair) - See time on X axis - Different lines = different doses of UV radaition Result - within 10 minuts the gene is induced = very rapid response system

Question 61

DNA damage signaling pathway in mammals

Answer 61

Humans - DNA damage response pathways are more complex Have a series of sensors --> feed into 2 larje protein kinases ATM and ATR --> ATM and ATR integrate damage and signals feed out to signals of resonse --> Cell cycle stops to stop dividing to fix DNA + regulates genes involoved in DNA damage + ca induce apoptosis if damage is bad + induce a series of stress resonse genes

Question 62

Recignition of single stranded breaks

Answer 62

Recognition of ssDNA breaks and ssDNA = uses PARP - Have an anticancer drug that targets this pathway (targets step between PARP bidning and Parylation) Start = have BER PARP recignizes ssDNA (acts as a sensor) -- PARP forms long chains of PolyADP ribose --> PARP ligates onto self and other proteins -> ligation serves as signal to repair single stranded break (recruits repair enzymes) Once repaired PARG = takes off the PolyADP ribose and restores to a normal state

Question 63

Homologous recombination and Homology directed repair

Answer 63

Homologous recombination and Homology directed repair are mechanstically related by produce dfferent results - Homologou srecombination is needed for generation of genetic diveristy - IN prokryotes homologou srecombination can occur between plasmid, phage, and host chromsomes during growth + conjugation + transduction + transfermation - In Eukaryotes homogous recombination can occur between plasmid, viral, and host chromsome during mitosis + transformation + meiosis - Homologous recombination = used to fix point muttaions + used in recombination bteween chromosmes

Question 64

Homologous recombination example

Answer 64

Have A,B,C and a,b,c on the two chrosmome copies --> can use the information from the good (red) copy to fix the error in repair OR chnage an allele - BOTH in the black copy) --> produces a b on the black chrosmome (donated by the red chrosmome) OR can have recombination in teh middle where the two ends have been chnaged ALL organism have HR

Question 65

How does Homoogous recombination occur (overall)

Answer 65

Homologous recombination is based on formation and resolution of Holliday junctions Holliday juntion - formed by reciprical exchange strands between homolgous DNA duplexes - resoloved by RuvC in E.coli ; Resolved by GEN1 in Eukaryots

Question 66

How does Homoogous recombination occur

Answer 66

Start - have two chromosomes --> Have strand invasion (Black chrosmome ssDNA invased the red chromomes which pushed the red ssDNA out of the way and the black strand forms a BP with the lower Red strand) --> cut aacross the region on the right --> seal nick = resuls in single cross over (holliday junction intermediate with DNA exchnage between the two chrosmomes) --> Holiday intermedits is free floating --> take the bottom chrosmoe a/b and invert it (seen in step 3) --> unqinds the corss oer in the middle --> can then bend the A/B side 45 degreesand the a/b at 45 degrees (see on RIGHT image) --> Can then cut horizontally or vertically --> Can resolve the structure by claving and ligating

Question 67

Cutting Holliday junction

Answer 67

Cut Horzontally - A and B are on the same strend with a little red peice in middle - IF exchnaged region had a missmatch --> issmatch can now be correct by MMR Cut vertically - Have A/b and a/B = chromsomal exhange (Have black red duplex) - Had exhange of the ends

Question 68

Holiday Junction Result

Answer 68

holiday jinction fomration and branch migration form heteroduplex substrates for mismatch repair - Mismatch correction can result in gene conversion Start with 2 chrosomes --> form heteroduplex DNA in Holiday junction (REd and black make dsDNA) --> have diffferent outcomes of mismatch repair - Can see fate of the middle Red/Bacl heteroduplex region --> BB pr bb or Bb or bB

Question 69

Possible outcome of homologous recombination

Answer 69

Homologous recombination in mitosis can resilt in loss of heterozygosity (LOH) - Rb is a tumor suprressor gene --> the cell that receoves both muttant copies loses growth control --> leads to cancer

Question 70

When can recombination occur

Answer 70

Recombination can occur in miesis (shuffles genetics) BUT can also happen in mitoticaly somatic cells - Mitotic recobination is not common but not rare IN mitosis it oftn does not have a big afect becase mom and dad will still ve presnet in the same cell at the same time BUT if one chrosmome has a mutation in a tumor supressor gene then there is risk that mitotic recombination rsults in a cell that is homozygous for the mutatnt

Question 71

Tumor supressor gene

Answer 71

Gene that keeos cell proliferation under conrti

Question 72

Why are yo at risk for cancer is have one copy of mutation in tumor supressor gene

Answer 72

If have one cpy of mutation in tumor supressor gene then you are helathy BUT at risk for cancer You are high risk because mitotic recombination bteween chrosmomes can lead to cell progeny that is homozygous for the mutatnt

Question 73

Example Loss of heterozygosity

Answer 73

If have 2 versions of the same chrosmoes where dad copy has a mutation and mom copy does not Nromally the cell is healthy with one normal copy Start - unreplicated chromosomes --> have replication - now have replicated chromosomes --> have mitotc recombination bteween chrosomes arms causes the dad arm with the mutation to go to the mom chromsomes --> chrosmomes seperate to duaghter cells - One daughter cell gets two helathy copies - One daughter cell gets two unhealthy copies of the tumor supressor --> can become a cancer cell

Question 74

Spectrum of Electromagnetic radaition

Answer 74

We only see a small part of the electromagnetic spectrum - Longer wL is infared = lower energy - Shorter wL = UV and X ray (higher energy) UV energy can make and break carbon-carbon and carbon-nitrogen bonds Xray and gamma rays can fragment DNA

Question 75

Deinococcus radiodurans and Thermococcus gammatolerans

Answer 75

NOTE - energy absrobd from gamma rays and X-rays = Grays (Gy) - ChesX ray = 1 mGy ; 5 Gy is lethal in human ' 5- Gy is ;ethal in E.coli Deinococcus radiodurans and Thermococcus gammatolerans = two bacterial species with gamma radiation resistence When companies were irradaited meat to sterlizie it using X-rays and gamma rays they discovered bacterial species that is very resistnt to radation (Deinococcus radiodurans and Thermococcus gammatolerans) - Deinococcus radiodurans and Thermococcus gammatolerans can surviave >5,000 Gy Resistnce liked evoloved as an uninteded side efefct of extreme resistnce to dessication (dessictaion = drying) - Dessication for bacteria is a stresser where there is DNA damage that is repaired in te same way as radiation damage

Question 76

Pulse Feild Gel

Answer 76

Run Pulse feild Gel - resovles high Molcular weight DNA by pulsing the elctric feild (swicth between having the elctric feild in the upper left then lower left) - IF have a long peice of DNA the chnages in electric feild causes the DNA to chnage shape in gel (chnages shape each time feild chnages) = DNA seperated in gel based on weight DNA = 50,000 bases = ver large (PFGE can seperate 1 million BP)

Question 77

Studying Deinococcus radiodurans and Thermococcus gammatolerans repair system

Answer 77

Run Pulse feild Gel Cut whole bacterial genome with RE (RE cuts infrequteley) --> can see fragments of genom on Gel (lane C) After radaition the same numver of cells are run --> have a lot of DNA at the orgine and the rest is lower in gel BUT the DNA is low MW (BUT still see cells survuve) 30 min - 1 hour have the same trend with the low MW weight DNA at the bottom of the gel decreasing at ecah time point UNTIL 3 hours At 3 hours the whole genome is back together - Genome was smashed because of radaition BUT then the peices are put back togetehr by homologous recombination/repair (Bacteria have a potent HR system that can assmble genome)

Question 78

Homologous Recombiation (Summary)

Answer 78

1. HR is intiation from a single or souble strand break 2. HR occurs through generation and resolution of holiday junctions 3. HR generates biological diversity through creation of new combinations of pre-existing genetoc varaition 4. HR can cause loss of Heterozyfgosity

Question 79

Endegenous mutogenic system

Answer 79

Endogenous mutogenic system = APOBEC family of enzymes - Have 12 APOBEC znzymes in humans APOBEC enzymes = cause deaminatiion of cytosine (Done in DNA and RNA) Purpose - viral defense mechansim - The best thing to do for a cell infection with virus is to stop the viral infection and commit suicide = indices APOBEC as anti-viral resonse = causes mutation in viral genome and own genome = cell dies and have no progeny

Question 80

APOBEC

Answer 80

APOBEC = Apolipoprotein B mRNA editting enzume catylytic polypeptde Function: 1. catylyzes convertion of C--U in DNA or RNA - When occurs in DNA it causes G-U mismatch 2. Mechansim of viral defense - when viruses replicate in a cell with APOBECs, the viral genome acquires so many mutations that the virus dies - APOBEC expression is very tightly controlled in uninfected cells 3. Cancer genome sequencing reveals that many cancers have patterns of mutations characteristic of APOBEC action + cancers over-express​ one or more APOBECs (pro-mutergenic thing occuring in cancer cells) - Mutations in cancer are the kinds that are dirven by APOBEC driven mutogensis

Question 81

APOBEC in human genome

Answer 81

Human genome codes for 11 APOBEC enzymes Example - AID (Activation induces cytodine deaminsae) --> creates the somatic​ mutations in B-lymphocytes that cause antibody genes to acquire mutations​ over weeks-months that lead to higher affinity antibodies

Question 82

Code for APOBEC

Answer 82

Message (mRNA) that codes for Apoipprotein B has a nucletode that is different from the sequence in the genome Have enzyme that comes in to find the mRNA and mutates the base = codes for a different Amino Acd - Enzyme converts C--> U in mRNA

Question 83

Regulation of APOBEC

Answer 83

Regulation of APOBEC is usually very repressed BUT regultion in not perfect --> can be taekn advantage of by cancer cells

Question 84

DNA damage and repair by numbers

Answer 84

Charts - shows the types of damage and teh events per cell per day Ex. Humans have 10,000 oxidation events and 10,000 depurinarion events per cell per day Body has and reapirs 10^17 DNA lesions per hour - 10^14 cells with MANY lesions per day