Lec 04- DNA Replication, Repair, and Recombination 2

Question 1

What are 2 main reasons DNA becomes damaged and needs repair?

Answer 1

• replication errors

- accidental lesions that occur in the genome

Question 2

How many accidental base changes result in a permanent mutation?

Answer 2

fewer than 1/1000 accidental base changes

Question 3

What is the spontaneous reaction that causes 5000 purine bases to be lost every day?

Answer 3

depurination

Question 4

What is the spontaneous reaction involving the conversion of C to U that occurs in 100 bases every day?

Answer 4

deamination

Question 5

What does exposure to reactive forms of oxygen in the cell or chemicals in the environment do to DNA?

Answer 5

causes DNA damage

Question 6

What does UV radiation exposure do to DNA?

Answer 6

produces a covalent linkage between 2 adjacent pyrimidies (pyrimidine dimers) such as T-T or C-T

Question 7

What happens when unprepared DNA is replicated?

Answer 7

deletion or a base pair substitution occurs in the daughter strand

Question 8

What are the 4 main types of DNA repair?

Answer 8

1) Base excision repair
2) Nucleotide excision repair
3) Transcription-coupled repair
4) Double-strand break repair

Question 9

What are the 2 types of Double-strand break repair?

Answer 9

• Non-homologous end joining

- Homologous recombination

Question 10

What types of DNA repair occur only on one strand?

Answer 10

• Base excision repair
• Nucleotide excision repair
• Transcription-coupled repair

Question 11

How many different DNA glycosylases are there?

Answer 11

at least 6 different types

Question 12

What is the function of DNA glycosylases?

Answer 12

each recognizes a specific type of altered base and catalyzes its removal

Question 13

What happens when the DNA glycoslyase flips out of the base from the helix?

Answer 13

it probes for damage

Question 14

What happens when the DNA glycoslyase finds an incorrect base?

Answer 14

it cleaves the glycosyl bond that connects the base with the sugar

Question 15

What do AP endonuclease and phosphodiesterase do in base excision repair?

Answer 15

cut the phosphodiester backbone

Question 16

What is the result of cutting the phosphodiester backbone during base excision repair?

Answer 16

• damage is removed

- gap is repaired

Question 17

When are depurinations repaired during base excision repair?

Answer 17

directly repaired beginning with AP endonuclease

Question 18

What are the steps of base excision repair?

Answer 18

1) DNA glycosylases probe for damage
2) If incorrect base, glycosyl bond between base and sugar is cleaved by specific DNA glycosylase
3) AP endonuclease and phosphodiesterase cut phosphodiester backbone (damage removed)
4) DNA pol adds new nucleotides
5) DNA ligase seals the nick

Question 19

How is nucleotide excision repair different from BER?

Answer 19

differ in how the damage is removed

Question 20

What kind of lesions can be repaired by nucleotide excision repair?

Answer 20

bulky lesions:

• chemically induced
• thymine dimers

Question 21

What does the multienzyme complex in the nucleotide excision repair do?

Answer 21

scans DNA for distortion in the double helix (instead of a specific base change)

Question 22

Where does nucleotide excision repair cleave the DNA?

Answer 22

cleaves the phosphodiester backbone on both sides

Question 23

What does DNA helicase do in nucleotide excision repair?

Answer 23

peels lesion-containing strand away

doesn’t remove any of the individual bases

Question 24

How is the large gap created by nucleotide excision repair fixed?

Answer 24

repaired by DNA pol and ligase

Question 25

How can cells preferentially direct DNA repair to sequences that are being actively transcribed (sequences that urgently need repair)?

Answer 25

by linking RNA pol with DNA repair

Question 26

What stalls lesions and directs repair machinery there in transcription-coupled repair?

Answer 26

RNA pol

Question 27

What does transcription-coupled repair work with to repair genes that are being expressed when the damage occurs?

Answer 27

BER NER others

Question 28

What is transcription-coupled repair specific for?

Answer 28

the strand being transcribed

Question 29

At what rate is the non-transcribed strand repaired in transcription-coupled repair?

Answer 29

non-transcribed strand is repaired at the same rate as the DNA not being transcribed

Question 30

What is cockayne's syndrome caused by?

Answer 30

defect in transcription-coupled repair

Question 31

What are some of the results from cockayne's syndrome?

Answer 31

- growth retardation - skeletal abnormalities - sensitivity to sunlight

Question 32

In Cockayne's syndrome, what does the defect in transcription-coupled repair do to the genes?

Answer 32

permanently stalls RNA pol at the sites of damage in important genes

Question 33

DNA molecules are __________ constructed for repair.

Answer 33

optimally

Question 34

Two strands of DNA allows a __________

Answer 34

backup copy

Question 35

What makes the distinction between damaged/undamaged DNA obvious?

Answer 35

the nature of the 4 bases

Question 36

What does every deamination event form?

Answer 36

an unnatural base | works as a signal

Question 37

What is a possible reason why RNA is not the hereditary information?

Answer 37

cannot distinguish between deaminated C and natural U

Question 38

What occurs at some CpG sequences?

Answer 38

special problem with methylated cytosines in vertebrate DNA

Question 39

What are associated with problems with methylated cytosines in vertebrate DNA?

Answer 39

inactive genes

Question 40

What does the deamination of methyl-C produce?

Answer 40

T mismatched with G

Question 41

What enzyme recognizes the mismatched T & G and removes the T?

Answer 41

DNA glycosylase

Question 42

The repair of T&G mismatch using DNA glycosylase is relatively __________.

Answer 42

ineffective

Question 43

What % of C nucleotides in the human genome are methylated?

Answer 43

3%

Question 44

How much do methylated C nucleotides account for all point mutations associated with inherited human diseases?

Answer 44

1/3 of all point mutations

Question 45

What are some of the causes of double-stranded breaks?

Answer 45

- ionizing radiation - replication errors - oxidizing agents - other metabolites

Question 46

What happens if double-strand breaks are left unpaired?

Answer 46

chromosomes would break into smaller fragments and be lost

Question 47

What process brings broken ends of DNA together and rejoins them using DNA ligation in a double-strand break repair?

Answer 47

Non-homologous end joining

Question 48

What occurs as a result of the process of non-homologous end joining?

Answer 48

1 or more nucleotides will be lost

Question 49

Why does the double-strand break repair process predominate in humans?

Answer 49

because so little of the genome is protein coding

Question 50

How many DNA scars will a typical 70 year old have from double-strand break repair?

Answer 50

2000

Question 51

What are checkpoints?

Answer 51

- an additional layer of regulation | - ensures the completion of one stage in the cell cycle before the next stage can begin

Question 52

What triggers checkpoints?

Answer 52

presence of DNA damage

Question 53

What are some of the checkpoints?

Answer 53

- blocking entry from G1-->S phase - slowing down progression through S phase - blocking transition from G2-->M phase

Question 54

Checkpoints give the cell ____________ to repair DNA damage

Answer 54

extra time

Question 55

What is homologous recombination?

Answer 55

genetic exchange between a pair of homologous DNA sequences

Question 56

What are some of the functions of homologous recombination?

Answer 56

- repair of double-strand breaks (at stalled or broken replication forks) - exchange of genetic information to create new combinations of genetic sequences (crossing over and gene conversion in meiosis)

Question 57

What mechanical role does homologous recombination play?

Answer 57

assuring accurate chromosome segregation

Question 58

What is the fundamental process of genetic exchange that is common to all organisms?

Answer 58

homologous recombination

Question 59

What is the location of homologous recombination repair?

Answer 59

at stalled or broken replication fork

Question 60

What kind of sequences does HR repair take place between?

Answer 60

similar sequences

Question 61

What does the process of HR require?

Answer 61

base pairing but it doesn't have to be a perfect match

Question 62

When a nick is encountered in the replication fork during HR, what will happen?

Answer 62

the replication fork will collapse and break

Question 63

What enzyme chews back the parental strand to prepare for strand invasion in HR repair?

Answer 63

5' exonuclease

Question 64

In HR, what is strand invasion?

Answer 64

pairing of single-stranded DNA with complementary strand in a different double-stranded helix

Question 65

What does strand invasion form?

Answer 65

forms a region of heteroduplex DNA

Question 66

What happens after strand invasion in HR?

Answer 66

- strands break and dissociate - DNA synthesis continues - replication fork restarts

Question 67

What guides homologous recombination?

Answer 67

base-pairing

Question 68

What do DNA duplexes do in HR?

Answer 68

sample each other to look for regions of homology

Question 69

What is hybridization?

Answer 69

DNA double helix reforming from its separated single strands "zipper"

Question 70

What is another name for hybridization?

Answer 70

renaturation

Question 71

What happens during HR once a region of homology is found?

Answer 71

the single strands rapidly pair up

Question 72

What is the result of hybridization?

Answer 72

creates a double helix from strands that originate from different molecules heteroduplex DNA

Question 73

What is heteroduplex DNA?

Answer 73

a double helix with strands that originate from different molecules

Question 74

What does DNA hybridization require?

Answer 74

a single-stranded DNA freed from pairing with complement so that it can pair with the second strand

Question 75

What directs the single-stranded invading strand?

Answer 75

- RecA (PRO) - Rad51 (EUK) - other proteins

Question 76

What is the DNA synapsis reaction?

Answer 76

- binds cooperatively to single stranded DNA | - holds it together with the double helix until homologous sequence is found

Question 77

How does the single strand search for the homologous sequence?

Answer 77

via an unknown mechanism

Question 78

What happens once a homologous sequence is identified?

Answer 78

- strand invasion occurs | - heteroduplex is formed

Question 79

What does the point of exchange move through once strand invasion occurs?

Answer 79

branch migration

Question 80

What happens during branch migration?

Answer 80

unpaired region of one single strand displaces a paired region on another single strand

Question 81

Branch migration characteristics

Answer 81

- happens spontaneously - both directions - catalyzed by special helicase to move in one direction (requires energy)

Question 82

How is non-homologous end joining different from homologous recombination?

Answer 82

Non-homologous end joining: - no template required - creates mutation at site of repair - can create translocation Homologous recombination: - uses daughter DNA duplex as template - no loss/alteration of repair site DNA - can repair other DNA damage types (versatile) - mechanism and proteins conserved in all organisms

Question 83

In repair of double stranded breaks by HR, what are the 5' ends degraded by?

Answer 83

exonuclease

Question 84

What invades the homologous template and primes repair DNA synthesis?

Answer 84

one 3' end

Question 85

What strand can anneal to the other original 3' overhang in the damaged chromosome through complementary base pairing?

Answer 85

the newly synthesized 3' end of the invading strand

Question 86

What does the use of a non-functioning homolog to repair the other homolog do in HR?

Answer 86

Causes loss of heterozygosity - critical first step in cancer development - rare occurrence

Question 87

How is repair prevented in the absence of damage?

Answer 87

- dispersed repair proteins throughout the cell | - repair occurs at the sites of damage in "factories" or "foci"

Question 88

BRCA2 maintains Rad51 (RecA) inactive. What happens if there is a mutation in BRCA2?

Answer 88

leads to increase of breast cancer

Question 89

To help demonstrate the localization of repair proteins to DNA damage, fibroblasts were x-rayed to produce ___________

Answer 89

double strand breaks

Question 90

What stain was used to show new DNA synthesis?

Answer 90

BrdU

Question 91

What does the Mre11 complex recognize in its localization to damage sites?

Answer 91

recognizes double-strand breaks and mobilized additional proteins to repair the damage