The Comet Assay. Heinz C. Base Excision Repair in Mammalian Cells. Mahmud K. Shivji, Jonathan G. Moggs, Isao Kuraoka, Richard D. Jill A. Mello, Jonathan G.
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Back Matter Pages About this book Introduction In this second edition of a much praised laboratory manual devoted to eukaryotic systems, Daryl S. Henderson has refocused the book on mammalian cells, adding fourteen entirely new chapters and extensively revising many of the remaining chapters. The authors address a broad range of questions about practical mammalian DNA repair, including such arcana as "what is radioresistant DNA synthesis and how is it measured?
The laser beam was scanned using a XY-scanning mirror introduced in the beam path before the telescopic lens system so as to create a line pattern across each nucleus. The focused laser-scanned area was estimated by multiplying the length of the line by the width i. The power of the laser beam at the sample plane was kept at 1.
The transmission factor of this objective was measured to be 0. The pulse energy at the focused spot was controlled by the orientation of the Glan-Thompson polarizer mounted on a motorized rotational stage. The exposure time at a focused spot was controlled by use of a computer-controlled mechanical shutter Uniblitz.
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The scanning pattern in the nucleus was generated by the scanning mirror, controlled by in-house developed software on a LabView platform and National Instrument's data acquisition and control board. A XY-scanning mirror was introduced in the beam path to enable scanning of the beam. The pulse energy at the focused spot was varied by a control on the orientation of the Glan-Thompson polarizer mounted on a motorized rotational stage. The scanning mirror was controlled by in-house developed software on a LabView platform to create a line pattern across the nucleus. The exposure time at a focused spot i.
The laser pulse energy or power at the object focal plane was determined by measuring the input energy at the back aperture of the objective multiplied by the transmission factor of the objective at that particular wavelength. In cases where the size of the beam was larger than that of the back aperture of the objective, an aperture with a diameter the same as that of the microscope objective back aperture was placed before the detector for power measurement.
HeLa cells were cultured and synchronized as previously described The staining procedure was described previously The experiments were repeated at least three times and each time six to seven cells were examined, which showed consistent results. S phase synchronization was done by double-thymidine block as previously described G1 phase cells were individually identified by following the mitotic cells as described 38 , In order to test whether other types of DNA damage are also induced resulting in activation of corresponding repair pathways, damaged cells were stained with antibodies specific for thymine dimers UV damage , specifically CPD and PP Figure 1.
The results of the ns green laser system were compared to other lasers, including ns UVA [high-dose relative to the dose required for DSB induction in the presence of BrdU see Table 1 ] without sensitization, fs NIR, and ps green laser systems. Comparison of threshold energies and specific parameters of five different laser microirradiation conditions necessary for induction of H2AX phosphorylation.
Corresponding brightfield phase contrast images are also shown. Factors involved in single-strand break SSB repair are recruited to the damage sites immediately following damage induction. The location of the induced lesions are indicated by arrowheads. Since the factors described above are also involved in BER, we tested for the presence of base damage in cells damaged by the three different lasers.viptarif.ru/wp-content/monitoring/4587.php
DNA Repair Protocols
Interestingly, base damage as represented by 8-oxoG was observed only with high-dose UVA, but not with either the green both ns and ps or NIR lasers Figure 1. In a previous report, the low-dose UVA laser system, which requires pre-sensitization of DNA with halogenated nucleotide analogs such as BrdU, failed to produce damage that allows visualization of NHEJ factor recruitment, such as Ku Using the low-dose UVA 0.
This is in contrast to the highly localized accumulation of Ku at the damage sites using the high-dose 0. NuMA, an abundant nuclear factor, failed to cluster to the damage sites, further supporting the specificity of the observed repair factor recruitment Supplementary Figure S3. Thus, depending on slight differences in the conditions, the same UVA laser can induce strong or impaired 53BP1 recruitment.
This emphasizes the need to clearly define the laser parameters employed. DSB responses induced by different laser systems. A Ku recruitment to the laser-induced damage sites. Immunostaining of cells damaged by different lasers as indicated using anti-Ku antibody. Lesions are indicated by arrowheads.
Corresponding brightfield phase contrast images for SA2 staining are also shown. Using the ns green laser, we previously reported the specific recruitment of cohesin to the damage sites, which was later confirmed by ChIP analysis of endonuclease-induced DSB sites 16 , 32 , 40 , Taken together with previous results by us and others 25 , 32 , 33 , 38 , green and NIR lasers are equally suitable for cytological observation of the recruitment of factors that do not form typical IRIF, while UVA without sensitization appears to cause aberrant DSB responses.
While threshold energy per pulse 0. The requirement for more pulses of the ns green laser compared to UVA may be in part due to the lower peak irradiance of ns green compared to UVA Table 1. Since the peak irradiance depends on the amount of energy in the pulse duration, the relative contribution of the various physical mechanisms underlying the induction of and type of DNA damage are also expected to depend on the pulse duration in addition to the wavelength.
Taken together, the results reveal that the efficiency of DSB induction is significantly different between different laser systems, indicative of different mechanisms by which damage is induced. We next compared the actual parameters used for our studies and others reported in the literature Table 2. Our UVA system is comparable to the condition used by Lan et al. This discrepancy may have led to significant under-estimation of the dosimetric values reported in their study This may be because of the larger focused spot size which led to lower peak irradiance in their system.
In contrast, nearly diffraction limited spot size as measured by lesion created on an air-dried red blood cell was achieved in our system using spatial frequency filtering of the laser beam by use of a pinhole placed in the beam path Table 2 ; [ 1 ] and [ 5 ]. While the study by Lan et al. However, it may be noted that the average power used for ns UVA laser is 0. The difference between our threshold and that of Lan et al.
Mikhailov et al. Their study had almost the same irradiance Table 2 ; [ 7 ] and [ 8 ] but less number of pulses 10 per spot to create DNA damage. The peak irradiance for our NIR laser Table 2 ; [ 11 ] is the highest of all the lasers used. In contrast, Mari et al.
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In the case of our system a significant opportunity for photothermal relaxation exists in between each macropulse that was not present in the study conducted by Mari et al. This may account for the dosimetry differences observed between our study and theirs. Our studies reveal that generation of different degrees of DSB, SSB, crosslinking, and base damage are dictated by different wavelengths, laser energies, pulse frequencies and durations and irradiances, indicating the involvement of different damage mechanisms. Surprisingly, the ns green laser, having similar peak irradiance, did not induce detectable PP.
DNA Polymerase β and Mammalian Base Excision Repair
This suggests that cumulative effects due to high repetition rate are required for PP production in the case of the green laser beam. Very little recruitment of endogenous XPC was detected under our conditions Table 2 ; [ 5 ] , while Lan et al. Taken together, the results indicate that unlike the conventional UVC-induced damage, no efficient activation of the NER pathway is associated with damage induced by ns UVA, ns and ps green, and fs NIR despite the presence of detectable crosslinking damage.
This may be because the amount of, or possibly the quality of, UV damage induced by these lasers is insufficient to trigger a significant NER repair response compared to the highly effective induction of SSB and DSB responses. Free radicals and reactive oxygen species ROS can be formed by various photochemical processes via linear or two-photon absorption. This is consistent with our observation that significant base damage is induced by UVA, but not by green or NIR lasers.
Frontiers | Comet assay to measure DNA repair: approach and applications | Genetics
Importantly, the results indicate that induction of a significant amount of base damage and abnormal DSB responses by UVA Figures 1 and 3 25 is wavelength-specific and cannot be attributed simply to the laser dose i. In the former case, the occurrence of DSBs depends on the efficiency of the incorporation of the sensitizing agents into both strands of DNA in close proximity otherwise resulting in SSBs. Hoechst non-specifically binds to the minor groove of DNA and may induce higher number of DSBs in the irradiated area.
Despite the large number of DSBs that efficiently recruit Ku, however, we failed to detect any measurable DSBs by the in situ comet assay 53 following ns green laser damage induction, suggesting that damage is clustered to such a small area, without scattering, that it is below the detection sensitivity of the assay data not shown. The cell viability and normal checkpoint responses in these cells also indicate that highly clustered DSBs, despite the large number, appear to have a less deleterious effect than widely scattered damage.
Thus, the biological effect of radiation dose and the number of DSBs are not necessarily comparable between laser microirradiation and conventional nucleus-wide ionizing radiation. Studies by us and others using the ns green and NIR lasers demonstrated that the factors involved in HR repair, such as Rad51, RPA and Rad54, are recruited to the damage sites even in the G1 phase of the cell cycle 37 , This can also be explained by the different densities of DSBs at the irradiated regions. It is possible that a high concentration of DSBs induced by green and NIR lasers results in the strong initial recruitment that may negate the necessity to form IRIF, which is secondary to the initial recruitment and requires distinct steps and factors In general, four potential mechanisms for the production of damage to the target structure can be considered: i temperature rise produced by linear or two-photon absorption; ii generation of large thermo-elastic stresses; iii various photochemical processes by linear or two-photon absorption including crosslinking damage and production of free radicals and ROS; and iv optical breakdown plasma formation produced by a combination of multiphoton and cascade ionization processes, leading to thermal, mechanical and chemical damage Figure 4.
Mechanisms of DNA damage by different laser microbeam systems. Three possible damage mechanisms single-photon absorption, multi-photon absorption, and plasma formation and their associated thermal, chemical and mechanical effects are listed. Although the thermal effect is most likely an important mechanism of damage induction, it is highly restricted temporally and spatially, and is dispersed instantaneously, and therefore any heat-inflicted damage outside of the focused area or any temperature rise in the cell is not expected to occur under the conditions used.
However, it is important to note that the temperature rise is proportional to the incident radiant exposure and if the spot size at the sample plane is not exactly diffraction limited as is the case for the nitrogen UVA lasers with non-Gaussian profile , the incident radiant exposure and therefore actual temperature rise can be an order of magnitude smaller. Furthermore, this high temperature will be dissipated over time and space.
Fig 5. Supporting Information. S1 Fig. S2 Fig. S3 Fig. Characterization of PARP-1 cleavage products. S4 Fig. S5 Fig.