Replication, Maintenance and Rearrangemnt of Genomic DNA part 2: DNA repair Flashcards
How can DNA damage occur?
- Spontaneous damage from the loss of the base or parts that results from exposure to harsh conditions (exposure to UV light or chemicals)
Give some examples of DNA damage induced by radiation and chemicals
- UV light: the formation of chemical adducts Thymine dimers
- Methyl, ethyl: Alkylation of Guanine when a methyl or ethyl group is added to the C6 of a purine ring
- Carcinogens addition of bulky chemcial groups: often carcinogen, can form complexes that will impede replication
Explain how the repair of O6-Methylguanine occurs.
- removal of CH3 from methyl guanosine is catalyzed by the enzyme O6-methylguanine methytransferase.
- the CH3 group is transferred to a cysteine and it becomes methycysteine
Explain the steps to Base-excision repair
- Removal of mismatch: DNA glycosylase catalyzes the removal of the base
- AP endonuclease cleaves next to apurinic or apyrimidic site: the AP endonuclease (AP for Apuric or Apyrimidic site) cleaves at the AP site
- Deoxyribose removal: deoxyribose is removed by the dexoyribosephosphodiesterase leaving a gap
- Correct base added by DNA polymerase: the gate will be filled by **DNA polymerase **
- phosphate backbone will be regenerated by DNA ligase (requires ATP)
Explain the direct repair of thymine dimers.
- photolyase: photoactivated enzyme that will cleave the dimer
- DNA polymerase: replaces the missing deoxynucleotides
other mechanism can do the same thing (Nucleotide Excision Repair NER)
Explain Nucleotide Excision Repair of thymine dimers
- Cleavage 5’ and 3’: NER requires cleavage of the damaged region a few bp upstream and a few bp downstream
- Unwinding by helicase: to separate the two strands DNA helicase will unwind the DNA helping to release the damaged fragment
- Filling by DNA polymerase and sealing the phosphate backbone by ligase
Explain NER in E. coli
uvrA, B, and C are linked to sensitivity to UV light
uvrA recognizes damed DNA and recruits uvrB and C then cleaves
the complex uvrA, B and C is also called exinuclease
Explain NER in yeast and higher eukaryotes
NER in yeast also uses UV-light damage repair system and the genes are called RAD
NER in higher eukaryotes: genes identified from patients with Xeroderma Pigmentosum and Cockayne Syndrome
A Large number of XP proteins have homologues in the yeast RAD genes
Summarize Nucleotide Excision Repair in mammalians
NER shares components with transcription machinery
- XP: Xeroderma Pigmentosum
- RPA: Replication Protein A
- ER: Excision Repair
- XP-B, XP-D: helicases
- XP-F, XP-G, ERCCI: endonuclease
- TFII H: helicase
- Gap filled by DNA pol
- Gap sealed by ligase
a. XPC (first) and hHR23B recognize DNA damage.
b. Recruit XPB and XPD (helicase) plus TF II H (helicase activity ).
c. XP G (endonuclease) added, TF II H maintains the region unwound
d. XP B and XP D maintain the DNA single stranded.
e. XP A, XP F and ERCC1 recruited.
f. XP F and ERCC1 acts as endonucleases.
g. The fragment is cleaved and released.
h. DNA pol fills the gap and DNA ligase reseals the DNA backbone
Explain transcription-coupled repair in mammalian cells
- Cockayne Syndrome patients are transcription-coupled repair deficient
- Strand specific repair linked with transcription
- Efficiency varies between transcribed and non-transcribed strand
- Uses XP proteins and ERCCI
- CS A and B refer to Cockayne Syndrome gene productss
- They are analogous to XP-C, hHR23B
- Another way to repair DNA is to take advantage of transcription separating the two DNA strands
- RNA polymerase stalls at site of damage
- CS-B is recruited and acts as a flag for addition of CS-A.
- CS-A and B are analogous to XP-C and hHR23B from NER
- The transcription coupled repair system shares components with NER and transcription machinery
- XP-B, D, F, G and ERCCI as well as TF II H are used
Explain mismatch repair in E. coli
a. Proteins MutS, L and H scan the newly synthesized DNA. Stop at mismatch, nicks the DNA strand (MutH). b. Mut H released.
c. MutS and MutL help recruit helicase and exonuclease.
d. the mutS protein recognizes and binds to mismatches in DNA duplexes.
e. The mutL protein binds to mutS.
f. The mutH protein binds to hemimethylated dam methylation sites.
g. The mutS-mutL-DNA complex stimulates mutH to cleave the unmethylated DNA strand at the GATC sequence.
h. The GATC site can be either 5’ or 3’ of the recognized mismatch.
i. One of two exonucleases (depending on whether cleavage was 5’ or 3’) chews away at the DNA to beyond the mismatch site.
j. Long patch repair synthesis follows.
k. Eukaryotes have proteins with sequence similarity to mutS and mutL that are involved in a similar repair pathway.
l. The eukaryotic mutS is a dimer of MSH2 and GTBP (now known as MSH3 or MSH6) proteins.
m. Eucaryotic mutL also consists of two polypeptides, MLH1 and PMS2.
n, The PMS2 gene was originally identified in yeast where its mutation causes abnormalities in post-meiotic segregation.
Explain mismatch repair in mamalian cells
MLH and MSH are eukaryotic homologs of MutS and MutL.
They bind mismatched bases and excise the DNA between the mismatch and nick in the DNA strand
The breaks in the Lagging strand are present at either end of Okazaki fragments
A break in the leading strand is present at the growing 3’ end
Explain transleasion DNA synthesis
- Was referred to as Error-prone repair
- In error-prone repair, the gap opposite the site of DNA damage is directly filled by newly synthesized DNA
- Because of damage to the template the repair is very inaccurate and leads to frequent mutations
- Error-prone repair is used only in bacteria htat have been subjected to protentially lethal conditions (such as extensive UV irradiation) where damage is so enormous that cell death is probably the only alternative
- Damaged DNA used as template
- Replication blocked by TT dimer
- A specific DNA polymerase (DNA V) can synthesize across a thymine dimer
- Eukaryote possess the same activity (9 enzymes known in humans)
- Those polymerase lack 3’ -5’ proofreaking activity that other DNA polymerases exhibit
Repair of double strand breaks
Ionizing radiation breaks the strands
Nucleotide removal, loss of bases and potential mutations
summary of cross talk in higer eukaryotes
