Cancer&dna Metabolism 1

Front 1

DNA Glycosylases

Back 1

Recognition of Damage specific Lesions, flip out mechanism, emzyme

Front 2

APEI

Back 2

Endonuclease, insition /tailoring of DNA backbone in BER

Front 3

Pol b (beta)

Back 3

Insertion of new nucleotide in BER

Front 4

Ligase III

Back 4

Ligation in BER

Front 5

NEIL 1/2/3

Back 5

Remove Base & Backbone &End?

Front 6

UNG

Back 6

Uracil glycosylase,

recognises U/A & U/G missmatches

Front 7

TDG

Back 7

Thymine glycosilase,

Recognises T/G missmatches

Front 8

OGG1

Back 8

Recognises oxidised G & removes it

Front 9

MUTYH

Back 9

After 1st replication with unnoticed oxidised G removes opposide A because it knows it shouldnt be paired with G

Front 10

Mule

Back 10

Ubiquinates pol B, regulation Pol B -> less polB

Front 11

ARF

Back 11

Produced if DNA has Damage, inactivates mule -> more pol B

Front 12

Things that make DNA unstable

Back 12

Cellulae metabolism

Hydrolysis

Alkylating Agents

(Endogenous&Exogenous sources)

Front 13

Type of damage repaired by BER

Back 13

Hydrolysis:Deputination,Depyrimidation

Oxidation: 8-oxo-Guanine, thymine Glycol

Nonenz. Methyl/alkylation :3-methy/adenine/guanine

Also Doublestrand breaks

Deamination: uracil

Aldehyde->backbone but no base

Front 14

BER ablauf

Back 14

1.Base recognition&removal (glycosylases) 2.incision or tailoring of DNA backbone (APE1&others) 3. insertion of nucleotide (polB) 4. ligation ligIII

Front 15

Hydrolytic Base Damage

Back 15

C ->U ,adenine->hypoxathine, guanine -> Xanthine, 5-methylcytosine->thymine(epigenetics)

Front 16

BER of Uracil

Back 16

With UNG

Shows no preference for U/A or U/G missmatches

Front 17

dUTPase

Back 17

Can also be falsly incooperated into DNA

U is incooperared opposide of A

Normal uracil missmatch U/G

Front 18

Muts/Mutsa (alpha)

Back 18

Recognition of missmatch & confromatial change

MMR

MSH2/MSH6

Front 19

MUTL/MUTLa (alpha)

Back 19

Recruited by MUTSa, strandspecific endonuclease, termination of exision

MMR

Front 20

RPA

Back 20

Protection of single stranded DNA

Front 21

EXO1

Back 21

digestion of Dna strand

Front 22

PCNA

Back 22

clamp of DNA by pol, activation MUTL, nicking of Daughter strand

Front 23

ligase 1

Back 23

ligation

MMR

Front 24

Benzo(a)pyrene diol epoxid

Back 24

Reacts with DNA-> Mutation von G/C zu T/A

Front 25

TSL pol

Back 25

more error prone Pol that gets switched in if normal pol cant continue

Front 26

BER of Tymine/Guanine missmatches

Back 26

with TDG-> T/G missmatch specific

dGTP icooperated opposide to T must not be adressed by TDG

TDG activity regulated during cellcycle

TDG in s-phase not around

Front 27

8-oxo-G

Back 27

führt zu prefered incooperation of A statt C

Front 28

2 sources of 8-oxo-G

Back 28

Ros oxidises G to 8-oxo-G-> CG:AT transversions

Ros ox. dGTP to 8-oxo-dGtp-> AT:CG transversions

Front 29

Regulation of BER

Back 29

Activity,Subcellular, Abundance

Front 30

Example regulation BER

Back 30

regulation of polB

with mule and ARF

Front 31

3 pols of replication&error rate

Back 31

(worst errorate)

Pol(alpha)->primer

pol(delta)->long strand

Pol(eta)->long strand ,leading strand

(best errorate)

Front 32

Errorate of pols composed of..

Back 32

1.Nucleotide selectivity

2.proofreading activity

(3.Missmatch repair?)

Front 33

Error rate of pols

Back 33

inrease of accuracy of 10^2 by proofreading

Replication accurate ~1error of 10^10 bases

Front 34

DNA lesions repaired by MMR

Back 34

-Missmatches->nur solange gepaart sind

-IDL`s

Front 35

Microsatelite instability

Back 35

diagnostic for impaired MMR repair

-> accumulation of errors as konsequenz of impaired MMR activity

Microsatellites are repeated sequences of DNA

Front 36

Proofreading vs MMR

Back 36

Proofreading -> primer not perfectly annealed

MMR-> primer perfectly annealed

Front 37

criteria for MMR

Back 37

1.MMR able to detect different missmatches

2.Repair before two strainds dissociate

3.Repair has to be directed to nascent strand

Front 38

MMR overview

Back 38

.... schau auf zf

Front 39

Benzo[a]pyrene diol epoxid

Back 39

Epoxide allows for reaction(convalent) with DNA

Disrupts Hydrogen bond

Pol weiss dann nicht was dahin kommt dann

MUtation from G/C->T/A

Front 40

DNA Pol switch

Back 40

-Führt zu mutagenesis

if normal pol cant continue more error prone pol TSL gets swiched in just to be able to continue.

after difficult part normalpol gets swiched in

Front 41

TSL

Back 41

errorprone Polimerase that gets swiched in

Front 42

UV ligth

Back 42

UVA->long waves

UVB

UVC->dosnt reach earth

Front 43

DNA lesions caused by UVA&UVB

Back 43

CPD&(6-4)Photoproduct

Front 44

CPD

Back 44

Cyclobutane pyrimidine dimer

Front 45

Why increase of scin cancer if people know is bad?

Back 45

-Uv light addictive -> evolutionnary so eigestellt

-keeps down certain autoimmune diseases&protection from them

-good vitain D status

Front 46

Photolyase

Back 46

repairs CPD's

needs visible light

dosnt exist in Humains ->got lost in evolution

exists in Gürteltieren

Front 47

Protection from uv damage in non Photolyasecreatures/ most mammals

Back 47

underground habitat, nocturnal behaviour,Fur,melanin,cellcycle checkpoint,immunesystem...

Front 48

NER &types of NER

Back 48

not dependent on visible light

GG-NER->Global genome NER

TC-NER->Transcription-coupled-NER

Front 49

NER DNA adducts removed as

Back 49

Oligonucleotide

Front 50

TC-NER& GG-NER ablauf

Back 50

siehe ZF

Front 51

XPC/CETN2/RAD23B

Back 51

Recognises Basepaar disortion opposide to damage

Front 52

UV-DDB

Back 52

Gets recruited& flips base out

recognises CPD&(4-6)PP

(4-6)PP->can also just get recognised by XPC because big distortion

Front 53

TFIIH

Back 53

Grosse ring um DNA -> 7 subunits

Front 54

XPD

Back 54

Helicase an TFIIH ring

is subunit of TFIIH

Front 55

RPA

Back 55

on single stranded DNA for protection

Front 56

XPF

Back 56

NER

incision on 3'

Front 57

XPG

Back 57

NER incision on 5'

Front 58

RFC

Back 58

loads PCNA for polimerisation

Front 59

PCNA

Back 59

clamp of DNA pol

important for polimerisation

Front 60

DNA Damage Response DDR

Back 60

Cellcycle checkpoints, DNArepair, Apoptosis

Front 61

Prot. Dynamics at sites of DNA breaks

Back 61

-Recruited: DNA Damage checkpoint factors,chromatin modulators,DNA repair factors

-Excluded: Transcription Maschinery

-Both directions: Chromatine components

Front 62

Sources of DSB

Back 62

-Exogenous agents: Ionizing radiation, chemicals(Topimerase inhibitors,crosslinking agents ICL)

-Endogenous causes: Spontaneous(ROS, Rep.fork collapse), Physiological induction(maiosis,V(D)J recombination)

-Experimental introduction: expression of DNA endonucleases(I-Seel, Cas9)

Front 63

TOP1

Back 63

Topimerase inhibitor

Generates DSB during DNA replication

eg. Camptothecin

Front 64

CTP

Back 64

Camptothecin

(TOP1)

CTP derivates used for cancer therapy(topocan&irinotecan)

Front 65

TOP2

Back 65

Topimerase inhibitor througout the cellcycle

eg. etoposide

Front 66

ICL

Back 66

DNA crosslinking agents

covalent crosslinks between bases

inhibits trancription or rep by inhibiting strand seperation

Front 67

Significance of DSB

Back 67

1.one unrepaired DBS can be enough to kill cell

2.Missrepair can lead to genomic instability&cancer

3. Inherited defects in DBS epair can cause cancer

Front 68

DBS detectors

Back 68

MRN complex

KU70-KU80-> Ku promotes NHEJ / reconnects two ends

Front 69

DBS repair Pathways

Back 69

Blunt ends-> c-NHEJ

sticky ends-> HR,SSA,ALT-EJ

Front 70

C-NHEJ

Back 70

classical non homologus end joining

Funcions in all cell phases

->accurate, 1~4 nt deletions

-Molecules: Ku70/80, DNA-PKCS,,DNA ligase IV XRCC4

Front 71

HR

Back 71

Homologous Recombination

Strand invation

only in S/G2 phase ->Stalls s-phase

->Accurate, LOH(loss of heterozygsity)

-Molecules:RPA,RAD51,BRCA2,,RAD51

Front 72

SSA

Back 72

Single Strand annealing

Annealing at long homologues

->Large Deletion,no insertions

-Molecules:RAD52,,DNA ligase I

Front 73

Alt-EJ

Back 73

Alternative end Joining

Annealing at microhomologies

->Mutagenesis,rearangement(IDL's)

-Molecules:PARP-1,,DNA ligase III

Front 74

Regulation of RAD51

Back 74

By regulating RAD51 ->regulate HR -> promote more errorprone alternative pathways like alt-EJ and SSA

Front 75

RAD51

Back 75

Important for invasion step in HR

Front 76

BRCA1&53BP1

Back 76

Antagonistic relationship

-beide vorhanden ode beide nockout ->Accurate DBS repair(HR& end processing)

-No BRCA1->Error prone DBS repair(no end processing,NHEJ)

Front 77

XLF-XRCCA ligase IV

Back 77

important for religation of both ends in c-NHEJ

Front 78

CtIP

Back 78

CtBP-interacting proteain

functions in cellcycle controll & transcript.regulation & DBS repair

localises DBS in S/G2 phase & key enzyme for DNA end resection(creation of 3' overhängs for HR)

Front 79

Sickness by mutations in CtIP

Back 79

Seckel & Jawad syndrome

neurological impairment

heterozygotes lower lifetimes

CtIP depleted cells hyper sensitive to drugs inducing DSB

Front 80

PARP inhibitors

Back 80

Makes that BER cannot function

PARP'S causes death in synthetic lethality only in cells that also have HR impairement (BRCA-)

->treatment for cancer

Front 81

CTIP is required for...

Back 81

-ssDNA formation in response to CTP treatment

-ssDNA formation in responseto IR

-recruitment of RPA to sites od DBS

-RPA foci formation

-RPA2 phsopharilat.(marker for resectedDBS)

-for HR

-for SDSA pathway?

Front 82

Disruption of CtIP affects DSB pathways

Back 82

-negatively: Alt-NHEJ,SSA,HR

-positif: total-NHEJ

->dh CtIP supresses c-NHEJ

Front 83

Evolutionary conservation of c-terminus of CtIP

Back 83

Always same terminus

Especially Base Thr at pos. 847

Thr 847 in humain gene is equivalent to Ser367 in yeast Sae2

Front 84

T847A Mutations

Back 84

impairs CtIP function

mutants sensitive to campthectin (CTP)

Front 85

Regulation of CtIP

Back 85

CtIP levels High in S phase and G2 pase

-regulated: post.transcriptionnal modification, protain-proteain interaction, proteain expression

Front 86

Ubiqutin system

Back 86

auf ZF

Front 87

End resection can be monitored by:

Back 87

-Brdu immunofluroscent staining

-RPA2 immunoblotting

(-Not gamma-H2AX immunofluorecent staining)