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Molecular Genetics > Repair Mechanisms > Flashcards

Repair Mechanisms Flashcards

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1
Q

how many repair genes are there in humans?

A

130

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2
Q

what are the five types of repair systems?

A
  1. direct reversal
  2. excision
  3. recombination repair
  4. nonhomologous end-joining
  5. synthesis of replacement DNA
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3
Q

what are the main two types of DNA damage?

A
  1. single base changes
  2. structural distortions
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4
Q

single base changes

A

affect the sequence but not structure; do not interfere with transcription or replication, hence potential mutations
- misincorporation
- deamination
- create mismatch that persists only until next replication, one copy is permanently mutated

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5
Q

single base distortions

A

impediment to replication or transcription
- thymine dimers by UV
- alkylation of G
- single-strand nick
- removal of a base

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6
Q

deamination

A
  • occurs with cytosine or guanine
  • removal of an amino group from a nucleotide base
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7
Q

alkylation of G

A
  • addition of methyl group
  • distorts the helix
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8
Q

depurination

A
  • bases are fragile and can fall of the backbone
  • only occurs with a G and A because they are puridines
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9
Q

deamination cytosine

A
  • deamination occurs, causes C to fall off
  • C replaced with U
  • corrected with mismatch repair
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10
Q

deamination of 5-methyl C

A
  • 5-methyl C is very common but can deaminate
  • C turns into T causing incorrect pairing
  • mismatch repair optimized for recognizing T-G
  • easily corrected
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11
Q

direct repair

A
  • rare in placental animals, common in bacteria and plants
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12
Q

excision repair

A
  • common way to repair, recognition enzymes sees damage, then excision of region that includes the damaged bases, followed by new DNA synthesis
  • often more bases are removed than just the damaged base
  • nucleotide
  • base
  • mismatch
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13
Q

nucleotide excision repair

A

removes sequence region with damaged nucleotide and synthesizes new DNA
- replaces a damaged strand

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14
Q

base excision repair

A

uracil glycosylase removes the base directly followed by replacement of a single base or a short stretch by DNA pol 1

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15
Q

mismatches

A

occur during replication and are corrected by distinguishing between old and new strands

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16
Q

photolyase

A
  • example of direct repair
  • uses light energy to break the crosslinks in thymine dimers
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17
Q

what is the process of nucleotide excision repair in E. coli?

A
  • recognition of mismatch and/or distortion of structure by endonuclease
  • incision: endonuclease cleaves both sides of damaged bases
  • excision: 5’ - 3’ exonuclease removes DNA between nicks
  • synthesis: DNA pol 1 replaces damaged region (DNA pol 3 used with mismatch repair)
  • ligation: ligase seals the nick
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18
Q

what is the Uvr system?

A
  • nucleotide excision repair
  • involved in short patch and long patch repair
  • long patch occurs 1% of the time and only under extreme mutagenesis is it used
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19
Q

Uvr A

A

recognizes distorted DNA
- this step does not use ATP

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20
Q

Uvr B

A

recruits UvrC and cuts
- melts the strands as well

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21
Q

Uvr C

A

cuts

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22
Q

Uvr D

A

helicase
- sometimes in this system an exonuclease can be used

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23
Q

how does the Uvr system repair DNA?

A
  • Uvr A recognizes distorted DNA while in a complex with Uvr B
  • A subunit burns ATP to disassociate from B
  • two C subunits attach to B and both cut 7 nucleotides on 5’ and 3-4 on 3’ (about 12) - this cutting requires ATP
  • Uvr D unwinds region to release strand, requiring ATP
  • DNA pol 1 synthesizes new DNA
  • ligase seals the nick
  • this is short patch repair and is used 99% of the time
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24
Q

long patch repair

A
  • triggered by SOS repair
  • pol 5 used
  • removes 1500 - 1900 nucleotides around replication forks
  • only used under extreme mutagenesis such as UV radiation
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25
Q

what is the Mfd system and how is it involved in transcription?

A
  • essentially Uvr in RNA
  • serves as a link between transcription and DNA repair
  • binds to stalled RNA polymerase
  • displaces RNA polymerase
  • recruits Uvr ABC excision repair proteins and directs repair of the template strand
  • Uvr does not initially detect damage but is directed to the site by Mfd
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26
Q

base excision repair: bacteria

A
  • deamination can be reversed by replacing U with C
  • consequence: UG replaces CG
  • uracil glycosylase corrects by cutting the bind between the base and the sugar
  • it is common for cytosine to spontaneously deaminate
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27
Q

recombination repair-retrieval system

A

corrects replication mistakes (gap in a daughter strand) by recombining with a good copy of the damaged region
- mostly found in bacteria

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28
Q

nonhomologous end joining

A

fixes double stranded breaks created by UV antibody shuffling

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29
Q

quality control

A

tolerance systems and error prone systems
- allow replication in case of structural damage accepting high error rate
- also important in eukaryotes

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30
Q

what are the major pathways of repair?

A
  • uvr
  • dam methyl directed replication mismatch repair
  • recBC and RecF
31
Q

eukaryotic excision repair pathway: transcription linked

A
  • TF2H attracted by a stalled RNA polymerase
  • RNA pol ubiquinated and tagged for degradation
  • TF2H has two transcription factors XPB and XPD
  • XPB melts the strands while XPD opens the bubble around the strands
  • XPG cleaves the 3’ side while XPF and ERCC1 cleaves the 5’ side
32
Q

what are the differences between global genome repair and transcription linked repair in eukaryotes?

A
  • global genome repair recognition complex XPC constantly scans and recognizes damaged DNA
  • the yeast homolog to XPC is RAD4
  • the XPG in global is related to FEN1
33
Q

what fills in the nucleotides in both transcription linked and global genome repair?

A
  • filled in by delta and epsilon polymerases
  • replaces about 25-30 nucleotides
34
Q

what is the process of Rad4-Rad 23 complex repair?

A
  • related to human XPC
  • complex flips the AA across from the TT dimers
  • Rad4 recognizes damaged DNA and recruits repair enzymes
  • a hairpin protrudes between strands at damaged region
  • recognition involves testing the stability of base paring
  • removes the As to allow work on the Ts
35
Q

how does Rad4 recognize damaged DNA?

A

correctly paired bases have tension that thymine dimers do not, which is what Rad 4 scans for

36
Q

what does mutations in XPB and XPD result in?

A
  • xeroderma pigmentosum
  • cockayane syndrome
37
Q

base excision repair: eukaryotes

A
  • directly removing a damaged base from DNA by glycosylase
  • lyase is a subunit of glycosylase that cuts the ribosugar, destabilizes the phosphodiester backbone and has a nick causing strand breakage and ensuring repair
38
Q

what occurs if lyase is not present?

A
  • if no lyase is present, APE 1 will perform the nick
  • APE1 will recruit delta and epsilon polymerase for long stretches
  • beta polymerase is used to replace short stretches
  • occurs immediately after replication
39
Q

which steps in the Uvr system do not require ATP?

A

recognition and binding of UvrAB to a mismatched base

40
Q

eukaryotic base repair: base flipping

A
  • base flipping is used by methylases and glycosylases
  • uracil and alkylated bases are recognized by glycosylases and removed directly from DNA
  • glycosylases scan the minor groove looking for damaged bases
  • pyrimidine dimers are reversed by breaking the covalent bonds between them
  • photolyase flips out the TT with the energy from light
  • yeast Ra 4 = pyrimidine dimer recognition, flips out the AAs on complementary strand leaving damaged TT exposed
  • methylase adds a methyl group to cytosine
41
Q

error prone repair

A
  • most repair systems are usually high fidelity
  • after UV radiation, error prone DNA replication can be induced
  • error prone phenotype may be part of a tolerance pathway, not just mutations in repair or replication genes
  • damaged DNA that has not been repaired causes DNA polymerase 3 to stall during replication and be substituted by Pol 5
  • DNA pol 5 can synthesize a complement to the damaged strand
42
Q

mutator phenotypes

A
  • mutations in the umuD and umuC inactivate DNA pol 5 and make UV irradiation lethal
  • these genes are part of the umuDC operon that is induced by UV related DNA damage
  • plasmids carrying homologs of these genes increase UV resistance to killing but the price is increase susceptibility to mutagenesis
43
Q

what is the activity once SOS repair is initiated

A
  • error prone activity umumDC operon encodes proteins that form the umuD’2C complex
  • umuD undergoes autoproteolysis by contact with activated RecA to form umuD’
  • the umuD’2C complex is called Dna pol 5
  • this DNA polymerase can bypass pyrimidine dimers or other bulky adducts and is only induced by the SOS system
44
Q

what is the purpose of mismatch repair?

A
  • removes errors that escape proofreading by DNA polymerase
  • mismatch repair increases the fidelity of DNA synthesis by 2-3 orders of magnitude
  • it must occur rapidly before the next round of replication to prevent a permanent mutation
  • it must be accurate by identifying the correct strand to change
45
Q

what are examples of mutator genes?

A
  • mismatch repair components
  • MutH, S, and L proteins
  • replication accuracy control components such as the epsilon subunit of polymerase 3
46
Q

when tow normal bases are mismatched, how does the cell know which base to change in order to restore the original state?

A
  • dam methylation system marks the original strand since it is fully methylated
  • in period before hemimethylated origin site is fully methylated by dam methylase, new strand is targeted for repair
  • strands now indistinguishable to repair system
47
Q

what is the system used for dam methylation mismatch repair in e. coli?

A
  • MutS recognition
  • recruitsment of MutL
  • MutH binds to MutS/L complex making the DNA loop
  • recognition of the GATC site causes MutH to join the complex where it acts as an endonuclease to nick the unmethylated strand
  • the mutation can be up to 1 kb away from the GATC site
  • UvrD helicase unwinds the nicked strand
  • 5’ to 3’ excision by exo 7 or 3’ to 5’ by exo 1
  • new strand synthesized by DNA pol 3
48
Q

what is the role of MutS?

A

recognizes the mismatch

49
Q

what is the role of MutL?

A
  • binds MutS and MutH
  • activated the endonuclease in MutH
  • recruits UvrD
  • binds beta clamp to recruit DNA pol 3
  • plays a key role in connecting mismatch recognition to strand identification and then recruiting the unwinding and repair of the excised region
  • most organisms do not have MutH, so MutL can have endonuclease activity
50
Q

what is the role of MutH?

A
  • endonuclease cuts 5’ to GATC on unmethylated strand
51
Q

how does MutS recognize a mismatch?

A
  • if the mismatch is present, a kink can be formed much more readily where correct base pairing is present
  • this causes MutS to bind ATP and adopt a conformation that allows it to slide along DNA
52
Q

most organisms, including bacteria, do not have Mut H homologs. How do these systems know which strand to correct for mismatched base pairs?

A
  • still an area of active research
  • MutS and MutL homologs bind to the mismatch
  • a helicase or nuclease enters the DNA at a nearby nick and cuts towards the mismatch and just beyond. DNA synthesis leads to repair
  • there are many breaks in the lagging strand immediately after replication that could be used to identify the new strand.
  • the leading strand has a single break. in eukaryotes, MutS/L is known to bind to the PNCA which could impart information regarding which strand is the newly synthesized one
53
Q

what does MutS have the highest affinity for?

A
  • has the highest affinity for GT mismatch
  • this results in efficient repair of GT to GC by the MutS/L/H system
  • mammals have a specific thymine glycosylase that removes T from a GT pair
54
Q

what repair affinity does MutY remove?

A
  • removes A from CA and GA mismatched pairs
  • does not use the MutS/L system
  • Mut encodes adenosine glycosylase which creates a apurinic site. this activates endonuclease followed by excision repair
55
Q

MutT

A

hydrolyzes 8-oco-dGTP
- hydrolysis of free nucleotide

56
Q

MutM

A
  • removes G=O paired with C
57
Q

MSH

A
  • yeast homolog to MutS/L
  • repair system in yeast that repairs mismatched base pairs
58
Q

Msh2

A

recognizes mismatch

59
Q

Msh3 an Msh6

A

specificity factors

60
Q

Msh2/Msh3 complex

A

binds insertion/deletion loops resulting from replication slippage

61
Q

Msh2/Msh6 complex

A

binds single base-mismatch
- do not use methylation state

62
Q

replication slippage

A
  • homologs in MutSL repair systems of eukaryotes
  • stuttering of DNA pol slips backwards and synthesizes extra repeating units seen as loops in sticking out of double helix
  • found to be defective in some human cancers such as colon
  • loss results in increased mutation rate
63
Q

what are the two rec pathways in retrieval systems?

A
  • RecBC and RecA
  • RecF, RecO, and RecR
64
Q

RecBC and RecA

A

used to restart stalled replication forks

65
Q

RecF, RecO, and RecR

A

used in repairing gaps due to TT dimers in daughter strand after replication
- RecOR and RecOF help RecA to form filaments on ss DNA even in presence of SSB
- RecBC and RecF gelp associate RecA with ss DNA

66
Q

recombination to recover replication errors

A
  • a replication fork may stall when it encounters a damaged site or a nick in DNA
  • a stalled fork may reverse by pairing between the two newly synthesized strands
  • a stalled fork may restart after repairing damage or use a helicase to move the fork forward
  • the structure of a stalled fork is the same as a holliday junction and may be converted to a duplex and ds break by resolvases
67
Q

what is the role of the RecA protein?

A
  • uvr and rec pathways are interconneced
  • uvr mutants, introduction of a mutation in recA eliminates all remaining repair capabilities
  • replication in uvr/RecA double mutants results in production of DNA fragments whose size corresponds to the distance between thymidine dimers; more than 1-3 thymidine dimers are lethal - wildtype can tolerate up to 50
68
Q

what are the activities of RecA?

A
  • the RecA protein is involved in normal recombination and in single-strand exchange of recombination-repair
  • the RecA protein is activated by UV irradiation and is thought to induce latent protease activity in its target proteins (LexA repressor and lambda repressor)
69
Q

what does damage to DNA cause?

A
  • causes RecA to trigger the SOS response
70
Q

what induction conditions are required for SOS response?

A
  • UV
  • cross linking and alkylating agents
  • thymine shortage
  • mutations in some dna genes
  • single-stranded DNA and ATP in vitro, same as required for RecA function in recombination
71
Q

what is the SOS system?

A
  • SOS repair system includes a by-pass system (tolerance system) that allows DNA replication across damaged areas at the cost of fidelity.
    Induced by inhibition in replication, UV damage, chemical crosslinking and alkylating agents.
  • includes long-patch excision repair and recombination repair pathways
72
Q

what are some theories regarding the mechanism of RecA activation?

A
  • DNA structure
  • small molecule released from DNA metabolism
  • single-stranded DNA and ATP
73
Q

regulation of the SOS system

A
  • LexA repressor protein normally keeps levels of LexA, RecA, and excision repair enzymes low
  • LexA is also induced but is rapidly inactivated by Rec A; activated RecA also causes autoproteolysis of umuD to form active DNA pol 4
  • there is a 50 fold induction of RecA
  • after damage is repaired, RecA is no longer activated. this results in the build-up of LexA protein, which shits down the SOS system
74
Q

why is LexA repressed by its own gene product?

A

it allows a rapid return to the non- error prone condition once the mutagen condition has dissipated

Molecular Genetics (28 decks)

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