BACKGROUND
Environmental chemicals can cause DNA damage, which also occurs naturally during regular cellular metabolism. Base damage and strand breakage are both unavoidable side effects of aerobic metabolism that are brought on by reactive oxygen species (ROS). The hydrolytic loss of bases, particularly purines, from the phosphodiester backbone, as well as the deamination and alkylation of bases, are additional spontaneous cellular processes.
According to estimates, each human cell produces up to 100,000 spontaneous DNA damages per day. The ultraviolet (UV) portion of sunlight produces cyclobutene pyrimidine dimers and oxidative base damage; ionizing radiation creates clusters of ROS that cause double-strand DNA breaks; and base-damaging chemicals like aflatoxins, benzo(a)pyrene, methyl chloride, and nitrosamines that alter or destroy DNA.
There is a need for a variety of very precise repair procedures because DNA damage has the potential to impede and/or change the fidelity of transcription and replication. Furthermore, required are bypass techniques that enable replication to proceed even in the presence of unrepaired damage.
In terms of the cognate lesions that each repair and bypass pathway may treat, there is significant overlap between them, according to an emerging theme over the past 20 years. Its functional redundancy emphasizes the significance of these pathways in the preservation of genome stability and is partially a result of the extremely high load of endogenous DNA damage.
Three distinct homologous recombination mechanisms have been identified in Saccharomyces cerevisiae: gene conversion, break-induced replication (BIR), and single-strand annealing (SSA), all of which rely on a collection of genes called the RAD52 epistasis group.
When DSB ends are cut by a 5-to-3 exonuclease, homologous recombination begins. This produces lengthy, 3-ended single-stranded DNA (ssDNA), which, in gene conversion or BIR, will invade a homologous donor sequence and serve as a primer for new DNA synthesis.
REQUIREMENTS
| Wild type Saccharomyces cerevisiae strain |
| Mutant Saccharomyces cerevisiae strain |
| Plasmid vector |
| Glycerol |
| U.V. light source |
| Replica plating block |
| Velvet cloth |
| Yest extract peptone dextrose medium (YEPD) |
| Peptone 20 g/L |
| Yest extract 10 g/L |
| Dextrose 20 g/L |
| Agar 15 g/l |
| pH 6.5 |
| Yest extract peptone galactose medium (YEP-GAL) |
| Peptone 20 g/L |
| Yest extract 10 g/L |
| Galactose 20 g/L |
| Agar 15 g/l |
| pH 6.5 |
| Yest extract peptone glycerol (YEP-Glycerol) |
| Peptone 20 g/L |
| Yest extract 10 g/L |
| Glycerol 38 ml/L |
| Synthetic complete (SC) medium |
| Yeast extract 6.7 g/L |
| Glucose 2% |
| Drop-out mix 2 g/L (contain all amino acids except uracil) |
| Agar 2% |
PROCEDURE
1. Select wild type strain of Saccharomyces cerevisiae and mutant strain of Saccharomyces cerevisiae (BJ2168 rad12) [1]
2. Select plasmid vector for yeast expression (e.g., pEGUh6, pEGTh6 etc.)
3. Both strain cells grown in a basic medium (YEP-glycerol) incubate at 30˚ C for 2-3 hr. [2]
4. Spread onto plates containing yeast extract peptone dextrose medium (YEPD) and yeast extract peptone galactose medium (YEP-GAL), incubate at 30-37˚ C for 24hr. [2]
5. By dividing the number of colonies growing on YEPGAL by the number of colonies growing on YEPD, the frequency of survival following a HO-induced DSB was calculated.
6. Cells resuspend into the YEPD medium with plasmid vector, incubate for 6-12 hr at 30˚ C.
7. Centrifuge the medium and collect cells of S. cerevisiae, prepare serial dilution of it.
8. By using appropriate dilution plate onto the YEPD plate.
9. YEPD plates irradiate with UV light for 30-60 sec. and keep one plate as control. [1]
10. Incubate irradiated plates in dark for 2-3 days at 30˚ C
11. By dividing colonies that survived UV treatment by colonies that did not get UV treatment, UV survival was determined.
12. To check repair analysis, the colonies growing on YEPGAL plates were replica plated onto the SC medium (without uracil) plates.
13. Check to see if colonies grown on SC media have repair mechanisms.
CONCLUSION
In this study, we used two types of Saccharomyces cerevisiae strains and checked their repair mechanisms by two methods: UV irradiation and replica plating on SC medium lacking uracil.
REFERENCES
- Zhongwen Xie, Shuqian Liu, Yanbin Zhang and Zhigang Wang, “Roles of Rad23 protein in yeast nucleotide excision repair”, Nucleic Acids Research, Vol. 32, No. 20, 2004.
- Jia-Lin Ma, Eun Mi Kim, James E. Haber, and Sang Eun Lee, “Yeast Mre11 and Rad1 Proteins Define a Ku-Independent Mechanism to Repair Double-Strand Breaks Lacking Overlapping End Sequences”, MOLECULAR AND CELLULAR BIOLOGY, Vol. 23 (23), December 2003.
- Serge Boiteux and Sue Jinks-Robertson, “DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae”, YEASTBOOK, GENOME ORGANIZATION & INTEGRITY, Vol. 193, April 2013.
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FAQs
Unlike BER, where the phosphodiester backbone is cleaved later, NER cleaves the phosphodiester backbone first.
Reversal damage, nucleotide excision repair, base excision repair, mismatch repair, recombination repair and error-prone repair.
Irradiation with U.V., IR, gamma rays or light, replica plating, PCR, electrophoresis, etc.
