ddPCR: Analyzing three DNA repair pathways together!

Parnad Basu, Amity University Kolkata

What is DSB?

DNA DSB (double-strand break) is a cytotoxic lesion. It is caused by ionizing radiation and radio mimetic chemicals. It can also be caused by a replicative DNA polymerase encountering a DNA single-strand break or by mechanical stress on chromosomes. Sometimes two complementary strands of a double-helical DNA break simultaneously. These breaking sites are close to one another. Which makes it impossible to keep the two DNA ends juxtaposed by the base pairing and chromatin structure. This is how DNA DSBs are generated. This can lead to genetic instability which may enhance the rate of cancer development.

DSB repair primarily occurs through 3 pathways:

1. NHEJ (non-homologous end-joining): It is the primary pathway for the DNA DSBs repair throughout the S (synthesis) and G2 (gap 2) phases of the cell cycle. NHEJ depends on Ku protein to repair the broken DNAs.
2. Alt-EJ (alternative end-joining): In the absence of Ku protein NHEJ cannot be performed. In that case, Alt-EJ repairs the broken DNA using microhomology. The key substance in Alt-EJ is DNA polymerase θ or Pol θ.
3. HR (homologous recombination): It comprises a series of interrelated pathways which help to repair DNA DSBs. HR includes the invasion of undamaged DNA by a damaged molecule of a similar sequence.

The insights into the recent study:

A group of scientists has developed a simple Cas9 (CRISPR associated protein 9) based system. This system can accurately represent the three pathways mentioned in the previous paragraph. It then converts it to ddPCR (droplet digital PCR) readouts which removes the need for Next Generation Sequencing and bioinformatic analysis. This ddPCR assay system gave some interesting insights. E.g., the absence of Pol θ repels only about 50% of total Alt-EJ. SSTR (single-strand template repair) needs only BRCA1 and MRE11 activity and not BRCA2 activity. This shows that the commonly used SSTR is not conventional HR. Also, Alt-EJ usage at double-ended DSBs is promoted by BRCA1 and not BRCA2.

How did they conduct the study?

Several controls were used by scientists to differentiate between NHEJ and Alt-EJ. For NHEJ, DNA-PKcs (DNA-dependent protein kinase, catalytic subunit) and LIG4 (DNA ligase 4) were used. Similarly in Alt-EJ, POLQ (DNA polymerase θ) was used. In the case of HR, MRE11i (double-strand break repair nuclease inhibitor) and CDK4/6 (cyclin-dependent kinase 4 and 6) were used as inhibitors.

At first the 250 ng (nanogram) genomic DNA was used in a PCR. It was then purified and Cas9 cuts were established using a T7 endonuclease assay. Scientists then did MiSeq sequencing. After which it was classified into three alignments. One, NHEJ (up to 5 bp deletion or 1 bp insertion). Two, Alt-EJ (more than 5 bp deletion). Lastly, HR (three 1 bp substitution). In ddPCR, a REF probe was always used. For NHEJ, Alt-EJ and HR 1 bp deletion, 12 bp deletion, and 3 bp substitution sequence-specific probes were designed respectively.

Results of the study:

1. Specific reads to differentiate between Alt-EJ and NHEJ were identified.
2. Supporting the Alt-EJ reads the 1 bp, 1 bp MH read reduced by 90% in a LIG4 knockout context. Whereas 12 bp, 5 bp MH read increased.
3. It provides the ability to apprehend multiple DSB repair pathways at a time.
4. Converting the reads to ddPCR readout makes the assay much more accessible.
5. Presented results help to clarify the role of BRCA1 in Alt-EJ. It was found that BRCA1 promotes Alt-EJ for a short-range deletion of 12 bp.

Limitations and conclusion:

Researchers noted mainly two limitations for this technique. One is that after NHEJ is repaired to the WT sequence, it cannot capture NHEJ repair reads. And the other is, HR repair events by the sister chromatid template couldn’t be measured. Once these limitations are solved, this new approach to DSB will be very important in the future.

Also read: Treating Irritable Bowel Syndrome with Low FODMAPs Diet

Reference:

1. Hussain, S. S., Majumdar, R., Moore, G. M., Narang, H., Buechelmaier, E. S., Bazil, M. J., Ravindran, P. T., Leeman, J. E., Li, Y., Jalan, M., Anderson, K. S., Farina, A., Soni, R., Mohibullah, N., Hamzic, E., Rong-Mullins, X., Sifuentes, C., Damerla, R. R., Viale, A., Powell, S. N., Higginson, D. S. (2021). Measuring nonhomologous end-joining, homologous recombination, and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell. Nucleic Acids Research. https://doi.org/10.1093/nar/gkab262

• The Corrosion Prediction from the Corrosion Product Performance
• Nitrogen Resilience in Waterlogged Soybean plants
• Cell Senescence in Type II Diabetes: Therapeutic Potential
• Transgene-Free Canker-Resistant Citrus sinensis with Cas12/RNP
• AI Literacy in Early Childhood Education: Challenges and Opportunities