lecture 13- mechanisms of DNA repair

Question 1

the most important cell function is to ___, this is done by ___

Answer 1

preserve genetic material (keep DNA integrity)
done by mechanisms of DNA repair

Question 2

name 6 mechanisms of DNA repair

Answer 2

mismatch repair
direct reversal of DNA damage
base excision repair (BER)
nucleotide excision repair (NER)
non-homologous end joining (NHEJ)
translesion DNA synthesis (TLS)

Question 3

mechanisms of DNA repair are the most ___ mechanisms of the cell because they use a lot of ___

Answer 3

costly
energy repairing DNA

Question 4

mismatch repair fixes ___

Answer 4

mismatched nucleotide replication errors

Question 5

mismatch repair involves the removal of several nucleotides from which strand?

Answer 5

the new strand

Question 6

briefly describe mismatch repair

Answer 6

usually “Mut” family enzymes- scan DNA, have to differentiate old parental DNA from new DNA, methylation– only parental (template for new DNA) is methylated, the enzyme knows the methylated enzyme is the correct one
- enzyme binds the mismatch, scans DNA bidirectionally, forms a loop…cleaves unmethylated DNA ahead of mismatch, helicase opens dsDNA and Pol I exonuclease degrades newly replicated DNA strand past the mismatch – Pol III fills gap – ligase seals DNA

Question 7

direct reversal (repair) of DNA damage corrects ___

Answer 7

damaged nucleotide base in one step

Question 8

name 2 examples of direct reversal of DNA damage

Answer 8

1- photoreactivation
2- removal of alkyl groups

Question 9

describe direct reversal of DNA damage in photoreactivation

Answer 9

repairs cyclobutane pyrimidine dimers (which form when DNA is under UV light)
- enzyme responsible for separating the dimer is DNA photolyase
- photolyase presents 2 chromophore groups, absorbs a photon of visible light and passes excitation energy (electrons) to FADH
- chromophore = group that absorbs light (transfers electrons)

Question 10

DNA photolyase uses the energy derived from ___ to reverse the damage caused by ___

Answer 10

absorbed visible light
UV light

Question 11

describe direct reversal of DNA damage- removal of alkyl groups

Answer 11

repair of oxidized nucleotides
- O6 methylguanine is repaired by enzyme O6methylguanine-DNA methyltransferase to become guanine again by catalyzing the transfer of the methyl group to one of the enzymes own cysteine residues
- enzyme is degraded after (fixes only one DNA alteration)
- remember O6 methylguanine pairs with thymine (transition)

Question 12

do photolyases repair pyrimidine dimers in humans?

Answer 12

no

Question 13

Base Excision Repair functions at the level of ___ and is involved in several types of repair mechanisms

Answer 13

single damaged nucleotide

Question 14

name 3 repair mechanisms by BER

Answer 14

1- removal of single damaged base (base was modified/damaged by alkylation, oxidation, or deamination); most uracils are removed by BER (deamination of cytosine forms uracils in DNA)
2- removal of abasic sugar (recognition and repair of AP sites- apurinic and apyrimidinic)
3- repairing single strand breaks that do not have the proper ends for ligase

Question 15

what recognizes the damaged base in BER?

Answer 15

DNA glycosylase

Question 16

describe BER in bacteria

Answer 16

formation of AP site — glycosylase cleaves base — AP endonuclease cleaves DNA and creates nick — 3’ OH and 5’ dRP — Pol I nick translation activity — ligase seals DNA

Question 17

describe BER in eukaryotes

Answer 17

2 paths for BER (long and short path)
- DNA glycosylase — AP endonuclease forming nick — in long path: Polymerase without 5’-3’ activity, need Flap Endonuclease (about 10 nuc’s fixed)
. in short path: Polymerase beta removes sugar phosphate from AP site (only 1 damaged nucleotide is fixed)

Question 18

BER in eukaryotes, ___ nuc’s are fixed in long patch and ___ in short patch

Answer 18

about 10
1

Question 19

glycosylases scan DNA and ___ …

Answer 19

flip the damaged base out of the helix and remove it if it fits in the catalytic site

Question 20

there are two main types of DNA glycosylases:

Answer 20

1- highly specific to a particular damaged base
2- recognizes oxidative damage in general (diverse substrates)

Question 21

Nucleotide Excision Repair target what?

Answer 21

large, bulky lesions and removes DNA on either side of them

Question 22

NER is the predominant repair pathway for removing _____

Answer 22

pyrimidine dimers, 6-4 photoproducts, and several other bulky base adducts, including benzo pyrene-guanine

Question 23

describe process of NER

Answer 23

• protein complexes recognize a variety of base damages resulting in distortions to DNA structure
• NER enzymes cleave damaged DNA strand on both sides of the lesion (removing more than the damage itself)
• specialized helicase “releases” cut fragment
• DNA polymerase fills in the single stranded gap using undamaged strand as a template
• ligase closes the nick

Question 24

unlike BER, NER does not…

Answer 24

require specific recognition of a damaged nucleotide

Question 25

NER in E. coli (bacteria) involves primarily 4 proteins:

Answer 25

UrvA, UrvB, UrvC, UrvD

Question 26

describe NER mechanism in bacteria

Answer 26

UrvA/UrvB scan for distortion in DNA –> UrvA then leaves, UrvB melts short stretch of DNA –> UrvC nicks both sides of distortion (about 12-13 bases) (in BER, 1 nick ; in NER, 2 nicks- 3’ and 5’) (UrvC called an exinuclease because it makes 2 nicks) –> UrvD (helicase- breaks H bonds) releases the fragment of DNA damage –> DNA Pol I fills gap, ligase closes gap

Question 27

NER in bacteria: function of UrvA

Answer 27

recognizes lesion

Question 28

NER in bacteria: function of UrvB

Answer 28

unwinds DNA

Question 29

NER in bacteria: function of UrvC

Answer 29

exinuclease- nicks on both sides

Question 30

NER in bacteria: function of UrvD

Answer 30

helicase- releases fragment of DNA damage

Question 31

NER in bacteria: function of Pol I

Answer 31

fills in gap

Question 32

NER in bacteria: function of ligase

Answer 32

seals DNA

Question 33

which repair mechanism fixes pyrimidine dimers in humans?

Answer 33

NER

Question 34

most of what we know about NER fixing pyrimidine dimers in humans is from research done on…

Answer 34

Xeroderma Pigmentosum- individuals extremely sensitive to sunlight
- mutations in at least seven genes (XPA-XPG) result in XP (defective NER)

Question 35

describe NER mechanism in eukaryotes

Answer 35

XPC recognizes lesion and recruits XPB & XPD (unwind DNA) –> they recruit XPF & XPG (XPF creates nick in 5’, XPG creates nick in 3’) –> RPA stabilizes the bubble –> PCNA is clamp loader that recruits poly’s to the rregion that fill gap –> ligase seals DNA

Question 36

NER mechanism in eukaryotes has global pathway, but also…

Answer 36

Transcription-coupled repair
- specifically targets repair to actively transcribed DNA
- when DNA being transcribed to RNA, RNA polymerase recognizes the damage and stalls transcription (RNA pol contains XPB & XPD) –> then continues on to XPF & XPG…

Question 37

double-strand breaks can be repaired by ___

Answer 37

homologous recombination (error “free”) typical route

Question 38

a repair mechanism for double strand breaks for phases of cell cycle when so sister chromatids are present is ___

Answer 38

non-homologous end joining (NHEJ)

Question 39

NHEJ usually leads to ___

Answer 39

mutations- loss of DNA

Question 40

briefly describe NHEJ

Answer 40

NHEJ present in all eukaryotes and some bacteria
- Ku70 & Ku80 bind to DNA ends in double strand breaks, they recruit other enzymes (DNA-PKcs and Artemis) - this complex fixes the double strand break
- XLF, XRCC4, and ligase IV seals the gap

Question 41

translesion DNA synthesis is repair for…

Answer 41

lesion at a replication fork after DNA strands have been unwound
- damaged DNA encountered at fork

Question 42

describe translesion DNA synthesis

Answer 42

DNA polymerase IV and V are used to extend DNA strand along template lesion —> DNA poly III comes again to continue replication