Mechanism of DNA double-strand break repair: Biochemical characterization of classical NHEJ and microhomology mediated alternative DNA end Joining.

Abstract

Maintenance of genomic integrity and stability is essential for organismal survival. DNA damage can occur in the form of base modifications, single-strand breaks (SSBs), and double-strand breaks (DSBs). Among these, DSBs are the most deleterious, potentially leading to chromosomal rearrangements and cancer if unrepaired or misrepaired.

Higher eukaryotes employ two major DSB repair pathways:

Homologous recombination (HR) - precise, active during S/G2 phases.

Non-homologous end joining (NHEJ) - error-prone, active throughout the cell cycle, and the predominant pathway in mammals.

Recently, alternative NHEJ (A-NHEJ) or microhomology-mediated end joining (MMEJ) has been identified, which operates when classical NHEJ (C-NHEJ) is compromised. MMEJ often uses short microhomology regions and is implicated in chromosomal translocations and cancer.

Classical NHEJ in Primary Tissues Cell-free extracts from rat tissues (brain, testis, thymus, spleen, lungs, heart, liver, kidney) were tested with DNA substrates mimicking endogenous DSBs.

Testis and lung extracts showed the highest repair efficiency.

Compatible and blunt ends were joined efficiently, while non-compatible ends showed minimal alterations.

Tissues with terminally differentiated cells (heart, kidney, liver) exhibited lower efficiency and more extensive deletions/insertions.

Protein expression analysis confirmed that tissues with higher repair efficiency expressed most NHEJ proteins.

Immunodepletion and inhibitor studies confirmed that KU70, KU80, DNA-PKcs, and LIGASE IV mediated the joining, establishing NHEJ as the major pathway across tissues.

Microhomology-Mediated End Joining (MMEJ) A novel biochemical assay was developed to study MMEJ using DNA substrates flanked by short homology regions.

MMEJ was slower than C-NHEJ and required lower protein-to-substrate ratios.

MMEJ was detectable even in normal cells, though less efficient than in cancer cells.

A minimum of 5 nucleotides of microhomology was required for efficient joining.

Sequencing revealed exonuclease digestion exposed microhomology, followed by alignment, flap deletion, and ligation.

Knockdown studies showed that KU70, KU80, and LIGASE IV were not required for MMEJ. Instead, MRE11, NBS1, LIGASE I, LIGASE III, XRCC1, and PARP1 were critical.

Heart Tissue as a Model for Alternative Repair Heart extracts showed negligible classical NHEJ activity but robust MMEJ.

MMEJ activity was detectable within 1 minute and optimal at 30 minutes.

Irradiation (5-20 Gy) increased MMEJ efficiency in heart extracts, accompanied by activation of pATM, XRCC1, and NBS1.

Immunostaining confirmed DNA damage response activation.