Retrieval System in E.coli .
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This system is often called post replication repair (or recombination repair). If there is a lesion in DNA (like pyrimidine dimer) which hasn’t been corrected, during replication, replicase won’t be able to make a complementary strand to the lesion on one strand. Replication will restart further downstream of the damaged parent strand.
NB one daughter strand is OK. The gap on the damaged daughter is filled by first stealing the strand from the good daughter. There is now a gap opposite a good copy and a good strand opposite the damaged base. Excision repair can now repair the problems.
It is the rec genes that perform this function. If a cell is excision- (uvr-) then if it is made RecA-, it cannot replicate its DNA properly.
Note the uvr and Rec functions are not independent.
RecA is involved in a cellular response to a comparatively large amount of damage, eg UV irradiation. They function partly to shut down some cellular processes to prevent damage to DNA or replication. The expression of many genes is involved. The response is basically an increased capacity to repair DNA by producing components of both long-patch excision systems and Rec recombination repair pathways. Cell division is inhibited.
RecA is activated by damage. A number of damaging effects can activate RecA, possibly a molecule released from damaged DNA. ssDNA and ATP is also required. The response is rapid.
Fig 14.32 in two parts.
LexA is also a repressor for RecA synthesis so its level goes up 50 fold from 1200 molecules/cel when it is cleavedl.
uvrB is one of the repair genes that is expressed. This, like other SOS induced genes has 2 promoters, so in presence lexA there is still a basal level of expression. LexA binds to SOS box.
The SOS pathway increases the levels of RecA and LexA (Does active RecA act as an enhancer of expression of LexA?).
The induction of RecA is required for its direct role in recombination repair and to cleave all of the LexA.
Once the inducing signal is removed, RecA can no longer cleave LexA, as it is being expressed at a high level and so quickly shuts down gene expression.
RecA cleaves UmuD, activating it and so the error prone repair system (long patch repair). RecA induces sulA which inhibits cell division.
Eukaryotic systems are harder to study. Can be studied by studying cell lines. Often rescuing mutation sensitive mutants to map the genes involved. A number of RAD genes (Radiation sensitive) have been found.
Rad3 group excision repair.
Rad6 group post replication repair.
Rad52 group recombination-like mechanisms.
By complementation of yeast mutants, human genes have been identified related to RAD3 and RAD10 (similar to E. coli UvrC).
Transcriptionally active genes are repaired preferentially. This involves a couple of subunits of a transcription factor TFIIH.
Xeroderma pimentosum (XP) a disease where the sufferer is extremely sensitive to UV. It is a failure in excision repair, cannot remove pyrimidine dimers. Six genes have been mapped with homology to RAD3.
Double strand breaks can occur. Homologous recombination can repair these breaks. This happens normally in the rearrangement of immunoglobulin genes, and sometimes due to irradiation damage.
The main mechanism to repair double strand breaks is by non-homologous end joining. This can result in the loss of bases. This would be an example of a tolerance system.
Repair of double-strand breaks by end-joining of nonhomologous DNAs (dark and light blue), that is, DNAs with dissimilar sequences at their ends.
Repair of double-strand breaks by end-joining of nonhomologous DNAs. These DNAs could be cut fragments from a single gene, or DNAs cut from different chromosomes. A complex of two proteins, Ku (70 + 80 dimer) and DNA-dependent protein kinase, binds to the ends of a double-strand break. After formation of a synapse in which the broken ends overlap, Ku unwinds the ends, by chance revealing short homologous sequences in the two DNAs, which base-pair to yield a region of microhomology.
The unpaired single-stranded 5′ ends are removed by mechanisms that are not well understood, and the two double-stranded molecules ligated together. As a result, the double-strand break is repaired, but several base pairs at the site of the break are removed. This can of course introduce mutations into coding genes. The majority in eukaryotes will not cause mutations as most of the DNA is non-coding.
Most eukaryotic cells are diploid. Homologous end joining can take place. This transfers intact genetic information from the intact chromosome to the site of the break. This requires recombination proteins and regions of DNA sequence match. In cells that have replicated but not divided this can repair exactly. This system is found more in prokaryotes and lower eukaryotes.
1 Why is this called post replication repair? At what point in the cell cycle does it take place?
2 What is required and what process is used to start the repair?
3 How is the repair completed?
In general mutants in any repair gene will have a higher frequency of mutation. Called mutator phenotype (Hence mut genes). They become very sensitive to any mutagenic agent, eg UV.
dam- mutants show increased spontaeous mutations as replication errors cannot be repaired. Also MutS, H or L.
Uvr-RecA- mutants abolishes all repair and recombination. When the nucleus replicates, there are many fragments of DNA due to the inability to replace thymidine dimers or repair the damage post replication.