The substrate of PARP is. . .
Creates a 5' single-stranded break at a site in the DNA double helix at an abasic site (presumably generated by DNA glycosylase).
DNA of all aerobically growing cells is exposed to reactive oxygen species during normal cellular metabolism
Mismatch repair mechanism
Role of p53 in the DNA Damage Response
Repair pathway overview
Creates a 3' break on an already 5' cleaved, abasic deoxyribose, freeing the deoxyribose and leaving a completely empty space on the DNA. Acts after APEI and DNA glycosylase in base excision repair.
Nonhomologous End Joining Mechanism
The first step of nonhomologous double strand break repair.
Recognizes breaks in the chromosome and brings in proximity, then recruits repair machinery.
Flips a residue targeted for base excision repair out of the double helix and cleaves off its base, leaving the phosphodiesters and deoxyribose intact.
Nonhomologous End Joining
Predominant mechanism of double strand break repair. Utilized in the early cell cycle and G0, before DNA has replicated.
DSBs are recognized by a protein complex, broken strands of DNA are aligned, and then re-ligated. Process is error prone as it does not depend on homology. Also has potential to lead to a chromosomal translocation.
Deamination of cytosine and adenine represent a very common mechanism of DNA damage via hydrolysis reactions
Direct repair mechanism
Simplest mechanism of DNA repair. An enzyme detects a base that has been damaged by addition of a functional group and removes the functional group. No further steps are taken.
Unfortunately, the utility of direct repair is limited to a small number of modifications.
Occurs when RNA polymerase is transcribing DNA and runs into a "bump" in the DNA that prevents it from moving. This leads to recruitment of the nucleotide excision repair complex, which repairs the lesion and allows transcription once more.
Mismatch repair always assumes that. . .
. . . the daughter strand is the one with the mistake.
CHK1 and CHK2
Induced by ATM and ATR. Arrest the cell cycle in two ways:
- Phosphorylate p53, preventing Mdm2-mediated export into the cytoplasm and subsequent ubiquitin-proteasomal degradation.
- Activate Wee1 and inhibit Cdc25, preventing the transition from G2 to M.
Base Excision Repair and Single-Stranded Break Repair Mechanism
Nucleotide excision repair
Removes larger lesions which are capable of distorting the overall structure of the DNA. This may include large DNA adducts spanning multiple bases or base dimerization catalyzed by UV light.
Mismatch repair can detect _____.
Mismatch repair can detect all four types of possible mismatch.
Master regulators of the DNA damage response
ATM and ATR
Both are protein kinases activated by MRN and KU (DNA breakage sensors), which in turn activate the distal transducers, CHK1 and CHK2, themselves kinases which induce p53-mediated cell cycle arrest.
an E3 ubiquitin ligase for p53
The cell's system for recognizing kinks in DNA
23B and XP-C, then RPA
Specificity in base excision repair relies on. . .
. . .the DNA glycosylases, which have evolved to recognize a number of different damaging DNA modifications.
Apurinic/apyrimidinic (AP) sites
Most frequent type of DNA damage. Occur spontaneously by hydrolytic loss of purine or pyrimidine at a given locus.
Poly-ADP ribose polymerase (PARP)
Recognizes a single stranded break in DNA (a missing residue on one side), and binds here. Generates Poly-ADP ribose at this site, a highly negatively charged polymer. This then recruits a repair complex, which removes the Poly-ADP ribose and attaches a DNA polymerase to fill in the single-nucleotide gap.
Repair mechanisms for double-stranded breaks
Homologous recombination is an error free mechanism, but this only works if there is a sister chromatid available to help repair the lesion (only in S or G2)
Nonhomologous end joining operates in G1 or G0. This can repair the DNA, but does so with much higher error rates.
Summary of Double-Stranded Break Repair Mechanisms
Homologous Recombination diagram
Pol β displays both ___ and ___.
Pol β displays both 5'→3' polymerase activity and 3'→5' exonuclease activity.
Thus it proofreads its last base added as it travels along the DNA polymerizing.
Weinberg-emphasized DNA damage → p53 induction pathway
XP-C and 23B
Recognize lesions that distort the shape of DNA. They then recruit TFIIH, a helicase that unwinds the DNA at this site. XP-G and RPA aid in the unwinding. Finally, XP-G and XP-F cut ~24-32 bases upstream and downstream of the lesion, creating a gap which will be filled in by Pol β.
Base Excision Repair
Most common mechanism of DNA repair. Recognizes a wide variety of modifications.
Detects a single damaged base pair and removes one base, along with part of its phosphodiester backbone. Then, the excised base is repaired using a single base which is complimentary to the base left behind. This leaves a single-stranded break, which is then repaired by common single-strand break repair mechanisms.
HMS-emphasized DNA damage → p53 induction pathway
Corrects mistakes made during replication. It is key here that this machinery is designed to remove the mismatched base on the daughter strand, NOT the template.
Predominant mechanism of double strand break repair utilized in S phase and later in the cell cycle. This mechanism is nearly error-free.
DSBs are recognized by a protein complex which resects DNA away from the DSB to make single-stranded regions (sticky ends). Once these are generated, resected strands invade the sister chromatid strands by identifying regions of homology. The sister chromatids are used as a template for new synthesis.
Basic DNA Repair Model
Nucleotide excision repair
Basic steps of DNA repair
- Remove the damaged DNA
- Fill the gap
- Ligate the DNA
O6-MeG vs N7-MeG
Futile cycles in nonfunctional DNA repair
caused by mutations in mismatch repair genes (MSH2, MSH6, MHL1), predisposes to development of colon cancer; 80% of patients carrying a mutation develop a tumor during their lifetime.
Mutations in BRCA1 or BRCA2 genes
involved in homologous recombination, predispose to breast and ovarian cancer; 30-80% of patients with a mutation will develop breast or ovarian cancer, depending on which gene is mutated
Mutations in XP
Xeroderma pigmentosum is due to mutation in nucleotide excision repair.
Direct repair enzymes tend to. . .
. . .steal and sequester modifications. They are not a type of enzyme which may replenish itself and continue to catalyze the reaction, it is a one-to-one ratio. Once the resource has been exhausted, mutations in DNA will accumulate.
Types of kinetochore attachment