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What needs to be repaired in DNA

replications errors
accidental lesions

1

Mutations doe not only occure because of mistakes in replication

spontaneous DNA damage

2

What is the spontaneous reaction to which 5000 purines are lost every day?

depurination
hydrolysis of the N-glycosyl linkage

3

What spontaneously occurs with 100 C to U bases/ day?

damination

4

What is caused by UV readation from the sun, or chemicals in the environment?

Produce a covalent linakge btw two adjacent pyrimidines (T-T) or (C-T)
purimidine dimers

5

What if DNA goes unrepaired?

deletion or base pair substitution in the duaghter strand

6

Base excision repair

Takes out the base, small repair

7

BER, six different types
- each recognizes a specific type of altered base and catalyzes its removal

DNA glycosylases

8

BER... Enzyme probes for damage by...

flipping out of base from helix to check if it is correct

9

BER, What happens if DNA glycosylases find an incorrect base?

cleaves glycosyl bond connecting base w sugar

10

BER What cuts the phosphodiester backbone to remove and repair gap?

AP endonuclease and phosphodiesterase

11

BER what are directly reapired begging w AP endonuclease?

depurinations

12

BER What adds new nucleotides/

DNA polymerase

13

What seals nick in DNA

DNA ligase

14

nucleotide excision repair...

repairs any bulky lesion like those chemically induced and thymine dimers

15

How does NER work?

a multienzyme complex scans DNA for distortion in double helix instead of specific base change

16

What does NER cleave?

phosphodiester backbone on both sides; DNA helicase peels lesion containg strand away

17

With NER what is the large gap repaired by?

DNA polymerase and ligase

18

Transcription coupled repair

cells can preferentially direct DNA repair to sequences that are being actively transcribed by linking RNA polymerase with DNA repair
- sequences that urgentyl need repair

19

TCR repairs by?

RNA plymerase stalls at lesions and directs repair machinery there

20

How does TCR work with BER, NER and others to repair genes?

Repairs genes that are being expressed when the damage occurs

21

What is TCR specific too?

the strand being transcribed

22

TCR

Non- transcribed strand repaired at the same rate as DNA not being trnascribed

23

-defect in transcritipon coupled repair
-growth retardation, skeletal abnormalitites, sensitivty to light
-RNA polymerase is permanetely stalled at sites of damage in importnat genes

Cockayne's syndrome

24

Damage detection

optimal for constructed remair
two strands=backup copy
all 4 bases are distinct so damage is obvious
RNA is not primary bc C and U would not be able to tell defect

25

special problem with methylated cytosines in vertebrate DNA

occurs at CpG sequences
associtated with inactive genes

26

deamination of methyl-C produces

T mismatched with G

27

what recognizes and removes the T

special DNA glycosylase
-repairs ineffectvie
-only 3% of C nucleotides in human genome are methylated, but it counts for 1/3 of all point mutaionts associated with inherited hjman diseases

28

Causes of double stranded breaks

ionizing, radiation, replication errors, oxidizing agents and other metabolites

29

if double stranded break is left un repaired

chromosomes would break into smaller fragments and be lost

30

how does DSB repair work?

non hologous end joining brings broken ends together and rejoins by DNA ligation; one or more nucleotides will be lost
-predominates in humnas and is genreally ok since so little of genome is protein coding

31

Damage delays the cell cycle

very importnat to maintian intact and undamaged DNA from one gnereation to the next

32

ensure the completion of one stage in the cell cycle before the next can begin

checkpoints

33

Presnend of DNA damage triggers various checkpoints

-blocks entry from G1 into S phase
- Slows down progression through S phase
-blocks transition from G2 to M phase
- goves the cell extra time to repair DNA damage

34

homologous recombination

genetic exchange btw a pair of homolgous DNA sequences

35

Repair of double stnrad breaks

homologous recombination
-especially at stalled or broked replicaiton forks

36

exhange of genetic informaiton to create new combinations of gnetic sequences

homologous recombination
-crossing over and gene converstion in meiosis

37

homologous recombination mechanical role

assuring accurate chromosome segregation

38

HR to repair stalled or broken replication fork

-exchange takes place btw similar sequences ... process requries base pairing but it doesnt have to be a perfect match
- replication fork will collapse and break when nick is encountered
-5' exonucleauses chews back parental strand to prepare for strand invation

39

pairs single standed DNA with complementary strand in different double standed helix
-forms a region of heteroduplex DNA

strand invasion

40

What happens in stand invasion

- strands break/dissociates
- dAN synthessi continues as replication fork restarts

41

What guides homolougus recombination?

base-pariing

42

HR

must be sequences that are similar

43

DNA dboule helix reforming from its spearated single strands

hybridizatino also called renaturation

44

once a region of homology is found,

the single strands rapidly pair up

45

creates a double helix from strands that orginate from different moclecules

heteroduplex DNA

46

What allows single stranded DNA pairing with homologous double helix

Rec A

47

What requires a single stranded DNA

DNA hybridization
-freed from pariing with complement so it can pair with the 2nd strand

48

How is the singld stranded invading strand directed?

RecA(Rad51 in eukaryotes) and other proteins

49

What happeds after the strand is guided to the SS

DNA synapsis reaction
-beinds cooperatively to SS DNA and holds it together with the double helix until homologous seqence is foudn
-SS searches via an unaknown mechanism

50

once homologous sequnce is identified...

strand invasion occurs, fomring heteroduplex

51

Once strand invations occurs, the point of exchang can move through?

Branch migration

52

Branch migration

unpaired region of one single strand displaces a paired region on the other
-happens spontaneously in both directions or can be catalyzed by special helicase to move in one direction

53

non-homologous end joinging

-no template requried
-creates a mutation at the site of repair
- can also create translocation

54

homologous recombination repair

-uses daught DNA duplex as template
-no loss or alteratioon of DNA at repair site
- can repair other tiypes of DNA damage
- very versatile
- mechanism and proteins conserved in all organisms

55

Repairing double stranded breaks by HR not at a replicaiton fork

- 5' ends degraded by exonucleases
- one 3' end invades homologous template and primes repair DNA synthesis
-gaps are filled in and ligated
-the newly synthesized 3' end of the inading strand is then able to anneal to the other orgiinal 3; overhang in the dmaged chromsome through complementary base pairing

56

homologous recombination in meiosis is slightly different

has many portine and sturcurual compononets in common

57

Regulation of homologous recombination

-accurate repair process can still casue problmes for a cell
-use of a non functioning homolog to repair the other homolog

58

What is a danger of homologous recombination?

loss of heterozygosity
-critcal first step in cancer development
-rare occurance

59

prevention of repair in the absnece of damage

repair proteins dispersed thoughout the cell
-after damage, repair occurs in factories or foci at the sites of damage

60

Regulation of homologous recombination of absence of damage

Brca2 maintains Rad51(recA) inactive
-mutations in BRAC2 leads to increase of breast cancer

61

localization of repair proteins to DNA damage

fibroblasts Xrayed to produce double strand breaks
-