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DNA Repair Mechanisms - Research Paper Example

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The DNA mechanisms of hyperthermophilic archaebacteria have been gaining interest because they must be able to work against the chromosomal damage that happens more often due to the extreme environment the organisms thrive at. In the research conducted by Rolfsmeier, Laughery and Haseltine (2010), the repair mechanisms of UV-C-induced double strand breaks in three strains of Sulfolobus solfataricus, a popular model system for hyperthermophilic bacteria, were characterized…
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Download file to see previous pages The optical density of the culture was maintained at 540 nm. The cultures were then exposed to different levels of UV-C, and were subsequently added to prewarmed ST medium. It was then incubated in the dark at 80°C with shaking. To determine which cells were viable, irradiated and control samples were grown in GT medium, diluted using the same medium, and plated in the dark on 0.8% (wt/vol) Gelrite (Kelco) GT plates with pH = 3.0. The plates were placed in a humid chamber with a temperature of 80°C for approximately 5 days, and colonies were counted. Meanwhile, growth rates were determined by spectrophotometric analysis at 540 nm of at least seven independent cultures grown in liquid, and generation times were calculated using Prizm 4.0 software. To visualize chromosomal damage, 4 x 109 suspended cells (optical density at 540 nm of 0.2 to 0.4) exposed to UV were obtained at 2-4 hours from the culture, and were treated with proteinase K and SfiI. The treated cell solutions were run using pulse-field gel electrophoresis (PFGE) with a buffer temperature of 14°C. with gels made of 1% agarose, 0.5xx TBE (9 mM Tris, 9 mM boric acid, 0.2 mM EDTA), and with running parameters of 24 h at 5.5 V/cm using a 120o included angle with switch times of 60 to 120 s. The gels were stained using ethidium bromide and, chromosome repair was measured using GeneTools quantification software, with the untreated sample designated as 100% repair. Finally, using PCR as well as gene and protein analysis, the sequences of the three strains were compared. Gene expression was then analyzed using RNA isolation, cDNA preparation, and qRT-PCR. The strains react differently to the varying amounts of UV irradiation. Upon the assessment of colony formation on solid medium, at the lowest UV dose (100 mJ/cm2), all three strains demonstrated resistance, with the highest survival rate recorded from strain 98/2 (23%, as compared to P2-A and P2-B, which had 11% and 13%, respectively). On the other hand, P2B was the most sensitive to 200 mJ/cm2, displaying 5- to 7-fold-lower survival than the other two strains, while 98/2 was the most sensitive at 300 mJ/cm2. Next, the growth rate after UV exposure was determined. Normally, and at 100 mJ/cm2, 98/2 grew the fastest, although exposure to UV, despite using the lowest dose, resulted to a decreased growth rate for all the strains. On the other hand, both P2A and P2B regrew faster than 98/2 after exposure to 200 and 300 mJ/cm2. Despite differences in growth rates, the growth levels achieved for negative controls achieved the same levels as the UV-exposed set-ups. Upon SfiI digestion and PFGE, inherent differences in the genomic sequences of these three strains were seen. In UV-protected set-ups, P2A, 98/2 and P2B had two, three and four distinct digest fragments, respectively, which means that there are sequence disparities among the strains. After irradiation, double-strand break formation, seen as lower molecular weight smears at the bottom of the gel, was present for all the strains, with most breaks and slowest acute (50%) repair rate observed from P2B. Overall, however, 98/2 ...Download file to see next pagesRead More
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