Repair Mechanisms Flashcards
how many repair genes are there in humans?
130
what are the five types of repair systems?
- direct reversal
- excision
- recombination repair
- nonhomologous end-joining
- synthesis of replacement DNA
what are the main two types of DNA damage?
- single base changes
- structural distortions
single base changes
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
single base distortions
impediment to replication or transcription
- thymine dimers by UV
- alkylation of G
- single-strand nick
- removal of a base
deamination
- occurs with cytosine or guanine
- removal of an amino group from a nucleotide base
alkylation of G
- addition of methyl group
- distorts the helix
depurination
- bases are fragile and can fall of the backbone
- only occurs with a G and A because they are puridines
deamination cytosine
- deamination occurs, causes C to fall off
- C replaced with U
- corrected with mismatch repair
deamination of 5-methyl C
- 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
direct repair
- rare in placental animals, common in bacteria and plants
excision repair
- 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
nucleotide excision repair
removes sequence region with damaged nucleotide and synthesizes new DNA
- replaces a damaged strand
base excision repair
uracil glycosylase removes the base directly followed by replacement of a single base or a short stretch by DNA pol 1
mismatches
occur during replication and are corrected by distinguishing between old and new strands
photolyase
- example of direct repair
- uses light energy to break the crosslinks in thymine dimers
what is the process of nucleotide excision repair in E. coli?
- 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
what is the Uvr system?
- 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
Uvr A
recognizes distorted DNA
- this step does not use ATP
Uvr B
recruits UvrC and cuts
- melts the strands as well
Uvr C
cuts
Uvr D
helicase
- sometimes in this system an exonuclease can be used
how does the Uvr system repair DNA?
- 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
long patch repair
- triggered by SOS repair
- pol 5 used
- removes 1500 - 1900 nucleotides around replication forks
- only used under extreme mutagenesis such as UV radiation
what is the Mfd system and how is it involved in transcription?
- 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
base excision repair: bacteria
- 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
recombination repair-retrieval system
corrects replication mistakes (gap in a daughter strand) by recombining with a good copy of the damaged region
- mostly found in bacteria
nonhomologous end joining
fixes double stranded breaks created by UV antibody shuffling
quality control
tolerance systems and error prone systems
- allow replication in case of structural damage accepting high error rate
- also important in eukaryotes
what are the major pathways of repair?
- uvr
- dam methyl directed replication mismatch repair
- recBC and RecF
eukaryotic excision repair pathway: transcription linked
- 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
what are the differences between global genome repair and transcription linked repair in eukaryotes?
- 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
what fills in the nucleotides in both transcription linked and global genome repair?
- filled in by delta and epsilon polymerases
- replaces about 25-30 nucleotides
what is the process of Rad4-Rad 23 complex repair?
- 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
how does Rad4 recognize damaged DNA?
correctly paired bases have tension that thymine dimers do not, which is what Rad 4 scans for
what does mutations in XPB and XPD result in?
- xeroderma pigmentosum
- cockayane syndrome
base excision repair: eukaryotes
- 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
what occurs if lyase is not present?
- 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
which steps in the Uvr system do not require ATP?
recognition and binding of UvrAB to a mismatched base
eukaryotic base repair: base flipping
- 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
error prone repair
- 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
mutator phenotypes
- 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
what is the activity once SOS repair is initiated
- 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
what is the purpose of mismatch repair?
- 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
what are examples of mutator genes?
- mismatch repair components
- MutH, S, and L proteins
- replication accuracy control components such as the epsilon subunit of polymerase 3
when tow normal bases are mismatched, how does the cell know which base to change in order to restore the original state?
- 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
what is the system used for dam methylation mismatch repair in e. coli?
- 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
what is the role of MutS?
recognizes the mismatch
what is the role of MutL?
- 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
what is the role of MutH?
- endonuclease cuts 5’ to GATC on unmethylated strand
how does MutS recognize a mismatch?
- 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
most organisms, including bacteria, do not have Mut H homologs. How do these systems know which strand to correct for mismatched base pairs?
- 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
what does MutS have the highest affinity for?
- 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
what repair affinity does MutY remove?
- 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
MutT
hydrolyzes 8-oco-dGTP
- hydrolysis of free nucleotide
MutM
- removes G=O paired with C
MSH
- yeast homolog to MutS/L
- repair system in yeast that repairs mismatched base pairs
Msh2
recognizes mismatch
Msh3 an Msh6
specificity factors
Msh2/Msh3 complex
binds insertion/deletion loops resulting from replication slippage
Msh2/Msh6 complex
binds single base-mismatch
- do not use methylation state
replication slippage
- 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
what are the two rec pathways in retrieval systems?
- RecBC and RecA
- RecF, RecO, and RecR
RecBC and RecA
used to restart stalled replication forks
RecF, RecO, and RecR
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
recombination to recover replication errors
- 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
what is the role of the RecA protein?
- 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
what are the activities of RecA?
- 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)
what does damage to DNA cause?
- causes RecA to trigger the SOS response
what induction conditions are required for SOS response?
- 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
what is the SOS system?
- 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
what are some theories regarding the mechanism of RecA activation?
- DNA structure
- small molecule released from DNA metabolism
- single-stranded DNA and ATP
regulation of the SOS system
- 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
why is LexA repressed by its own gene product?
it allows a rapid return to the non- error prone condition once the mutagen condition has dissipated
