Lecture 17 Flashcards
BRCA1
the product of the first gene implicated in familial breast and ovarian cancer susceptibility
ATM
plays a pivotal role in communicating DNA damage through signal transduction to activate transcription of the p53 gene, which codes for a TF. ATM activates the p53 repair pathway.
How does ATM-p53 repair pathway control cell responses to mutations?
in two ways:
- pause the cell cycle at the G1 to S transition to allow time for repair
- Direct the cell to undergo programmed cell death (apoptosis)
level of p53 in normal cells?
p53 levels are low in healthy cells (Mdm2-mediated degradation) but ATM increases the level in response to DNA damage. High p53 (phosphorylated) level initiates g1 arrest, this allows for time to repair damaged DNA. Completed repair depletes p53 and allows the cell cycle to proceed. If the p53-induced pause of the cell cycle persists too long (damage cannot be fully resolved), the apoptotic pathway is induced.
Translesion synthesis (TLS)
a DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymidine dimers or AP sites. It involves switching regular DNA polymerases for specialized translesion pols (i.e DNA pol IV or V in e.coli). These “error-prone” pols have no proofreading ability, and can replicate across lesions that would stall DNA pol III.
Nonhomologous end joining (NHEJ)
repairs ds breaks occurring before DNA replication. NHEJ inevitable leads to mutation since it is error-prone.
Process:
- After the ds break is produced, ds breaks are recognized by a protein complex (PKcs, Ku70m Ku80) that attaches to the broken ends of the DNA.
- The complex trims back the free ends of the breaks (resection). However, resection has removed nucleotides that cannot be replaced.
- Blunt ends produced by resection are rejoined by a specialized DNA ligase. Replication may now occur across the repaired region.
synthesis-dependent strand annealing (SDSA)
DS breaks that occur after replication can be repaired by this error-free process. Requires sister chromatid.
Process:
- SDSA begins with the trimming of one of the broken ds strands
- Followed by attachment of the protein Rad51. Rad51 facilitates the invasion of the intact sister chromatic by the resected end of the broken strand
- The strand invasion process displaces one strand of the intact duplex, forming a displacement (D) loop
- replication within the loop synthesizes new DNA from the intact template strand
- sister chromatids reform by dissociation and annealing of the nascent strands to repair the breaks, resulting in replacement of the excised DNA with a duplex identical to the sister chromatic
Homologous recombination
DS break initiates homologous recombination during meiosis. It is the exchange of genetic material b/w homologous DNA molecules.
- in bacteria, it occurs during events such as conjugation and as a consequence of a ds break repair
- in eukaryotes, homologous recombination takes place in prophase I of meiosis and is initiated by controlled ds DNA breaks
- DS breaks that initiate recombination are spontaneous, but are generated in a programmed manner by the activity of a specialized enzyme
Key molecules for Homologous recombination
- Spo11: eendonuclease (meiosis)
- Mrx complex (Mre11-Rad50-Nbs1): 5’ end resection
- Rad51/Rad54: strand 54
- Dmc1: strand invasion (meiosis)
Homologous recombination process
1) recombination is initiated by Spo11, it generates a ds cut in one chromatic.
2) proteins Mrx and Exo1 associate with spo11 and help with enzymatic digestion (5’ and 3) to generate ss segments
3-4) the proteins Rad51 and Dmc1 (homologs to e.coli RecA) help facilitate strand invasion and D loop formation.
5) the two strands that appear to cross over one another form a Holliday junction. There is also a heteroduplex region. The heteroduplex is ds DNA formed from DNA of different homologs. The heteroduplex may contain nucleotide mismatches (i.e heterozygotes alleles)
6) The D loop is “Captured” by the non-invading overhang. A second heteroduplex region forms. Extension of the broken strand and DNA synthesis are guided by the intact template strands and assisted by proteins including Rad52 and Rad59.
7) DNA pol strand extension and ligation fills gaps
8) the 3; end of the broken strand joins the 5’ end of the initial invading strand. Nonsister chromatids are now connected by double holliday junctions (DHJs). The resolution of DHJ requires strands to be cut and rejoined, such that the chromatids can separate. It is not clear how exactly the DHJ is resolved. DHJ can be resolved in two ways, to produce crossovers (opposite sense) or non crossovers (same-sense).
opposite sense resolution
involves cutting and rejoining of DNA strands in one of the Holliday junctions and cutting and rejoining of the two strands outside the second Holliday junction (flanking genes) . The result is recombinant chromosomes.
Same sense resolution
involves cutting and rejoining of the DNA strands in both holliday junctions. Genetic recombination does not take place (no flanking genes). Less common, thus homologous recombination in meiosis is likely to lead to production of recombinant chromosomes
Transposable genetic elements
DNA sequences that can move within the genome by an enzyme-driven process, transposition. They vary in length, sequence composition and copy number.
Movement occurs in two ways:
- nonreplicative transposition: Excision of the element from its original location and insertion in a new location
- replicative transposition: duplication of the element and insertion of the copy in a new location
DNA transposons
Class II transposable elements. Transpose as DNA sequences and may be replicative or nonreplicative.
Retrotransposons
Class I transposable elements. Are composed of DNA but transpose through an RNA intermediate. The RNA is copied back into DNA by reverse transcriptase. The reverse transcribed DNA is then inserted into a new location where flanking direct repeats are formed
