End Protection
Ku proteins and the 53BP1-associated regulatory network help protect DNA ends from extensive resection, supporting classical NHEJ.
EDITGENE / MOLECULAR REPAIR NETWORK / 004
← Genome Atlas
01 / The Molecular Breakpoint
One break.
Multiple
futures.
When both strands of DNA are broken, the cell must respond to preserve genetic information. Distinct molecular repair pathways can rejoin DNA ends, use short sequence homologies, or reconstruct information from a homologous template.
Explore the Repair NetworkFigure 01. A simplified representation of a DNA double-strand break. Different break-end configurations can activate different repair responses.
02 / Repair Pathway Cartography
Navigate the molecular repair network.
Explore three important repair strategies. Select a pathway below the branching map to change the molecular outcome illustration and view its associated mechanism.
Branch 01 / c-NHEJ
Rejoin the DNA ends.
Classical NHEJ uses end-binding, processing and ligation factors to reconnect broken DNA. Some breaks can be repaired without sequence alteration; others produce small insertions or deletions.
Associated factors
Ku70/80, DNA-PKcs, XRCC4,
DNA ligase IV and accessory factors.
Branch 02 / MMEJ
Align short shared sequences.
After DNA-end resection, short matching sequences can anneal, facilitating joining through alternative end-joining mechanisms. Polymerase theta is important in many theta-mediated end-joining reactions.
Typical outcome
Deletions between microhomologies,
sometimes accompanied by
templated insertions.
Branch 03 / HDR
Use a homologous template.
Homology-dependent repair uses matching DNA sequence information to restore or, in genome engineering contexts, introduce selected changes. Homologous recombination is particularly active when sister chromatids are available.
Important distinction
In genome editing, HDR often
describes homology-directed
incorporation of sequence
from a supplied donor template.
This network illustrates three important
DNA repair strategies, not all repair
pathways. Single-strand annealing (SSA),
additional HR subpathways and other
repair mechanisms also contribute.
Selecting a branch changes the explanation,
not the behavior of a real cell.
03 / Mechanism Atlas
Three strategies. Different molecular logic.
End joining, microhomology use and homology-directed repair differ in their molecular requirements and potential sequence outcomes.
I.
Classical Non-Homologous End Joining
Classical NHEJ protects and brings together broken DNA ends. The Ku70/80 complex recognizes the break and helps recruit DNA-PKcs and additional processing factors. DNA ligase IV, associated with XRCC4, completes joining.
Depending on end compatibility and processing, joining can preserve the original sequence or introduce small insertions and deletions. NHEJ operates throughout the cell cycle and is a prominent repair pathway in mammalian cells.
End protection
DNA ligase IV
Variable indels
May be sequence-preserving
II.
Microhomology-Mediated End Joining
MMEJ is a form of alternative end joining associated with DNA-end resection and short stretches of matching sequence. Resection exposes microhomologies, which can anneal before remaining overhangs are processed and the DNA ends are joined.
Polymerase theta supports many theta-mediated end-joining events. Because DNA between the aligned microhomologies can be lost, this mechanism is frequently associated with deletions. Templated insertions can also occur.
End resection
Short microhomologies
POLQ / Pol θ
Deletion-associated
III.
Homology-Directed Repair
Homology-directed repair uses homologous sequence information as a template. In homologous recombination, resection generates single-stranded DNA, while proteins including BRCA1, BRCA2 and RAD51 support template-directed repair processes.
Sister chromatids provide high-fidelity repair templates during parts of S and G2. Genome engineering strategies may instead seek to introduce an intended modification from a supplied donor template. These outcomes are not guaranteed and may compete with end joining.
Homologous template
RAD51
BRCA proteins
Cell-cycle dependence
04 / Repair Pathway Regulation
The proteins behind the repair decision.
Repair pathways depend on coordinated signaling and protein recruitment. The regulation of DNA-end resection is particularly important in repair pathway choice.
Figure 04. A conceptual regulatory network. The actual protein interactions are more complex and involve feedback, redundancy, additional repair pathways and cell-specific regulation.
End Resection
Factors including the MRN complex and CtIP contribute to the formation of single-stranded DNA intermediates associated with several other repair pathways.
Template and End Processing
Polymerase theta supports many MMEJ reactions, while RAD51-mediated strand exchange contributes to homologous recombination.
05 / Genome Editing Outcomes
Repair creates the final sequence.
In many nuclease-based genome editing experiments, DNA cleavage is only the initiating event. Cellular repair processes influence the observed genotype.
01
Sequence-Preserving Repair
Some DNA breaks can be rejoined without changing the original nucleotide sequence. This is one possible outcome of classical NHEJ.
02
Small Insertions & Deletions
Repair-associated indels can disrupt coding sequences or regulatory elements. The result is dependent on the target and the resulting sequence, not simply the presence of an indel.
03
Microhomology-Associated Deletions
MMEJ can align short repeated sequences exposed after resection. The intervening segment is often lost, creating characteristic deletion outcomes.
04
Template-Directed Sequence Changes
A homologous donor template can support incorporation of a selected sequence modification, although competing pathways and cell context influence its frequency.
05
Complex Genomic Alterations
Some editing experiments produce larger deletions, rearrangements or other structural changes that may not be visible in a short targeted amplicon.
Scientific interpretation:
a pathway preference does not guarantee
a particular repair outcome. DNA-end
structure, cell type, chromatin state,
target sequence and experimental
conditions all influence the result.
06 / Interpretation Laboratory
What does the repair evidence reveal?
Molecular characterization can identify repair products, but assigning every observed allele to one pathway is not always straightforward. Explore the questions that matter when interpreting repair outcomes.
01 How are repair products characterized?
Targeted sequencing can identify substitutions, short insertions and deletions around the investigated genomic locus. Different sequence products may appear in one cell population.
ANALYTICAL CONSIDERATION
Assay sensitivity, amplification biases and the ability to detect larger changes influence interpretation of repair outcomes.
02 Can sequence patterns reveal a pathway?
Microhomology-associated deletion patterns and small indel signatures may suggest contributions from particular repair mechanisms.
ANALYTICAL CONSIDERATION
Sequence signatures alone do not always identify the responsible pathway uniquely, because repair mechanisms can generate overlapping products.
03 Why does cell-cycle stage matter?
Homologous recombination is particularly supported when a replicated sister chromatid is available. NHEJ can operate across the cell cycle.
ANALYTICAL CONSIDERATION
Cell-cycle stage is one influential factor rather than a universal predictor of repair pathway use.
04 Why investigate genomic integrity?
Some repair products extend beyond the immediate cleavage site. Large deletions, rearrangements or unexpected integrations may require specialized characterization.
ANALYTICAL CONSIDERATION
Complementary assays, including suitable long-range or structural variant methods, may be needed to evaluate genomic integrity.
07 / Scientific Literature
Explore the science behind DNA repair.
Peer-reviewed reviews and research provide a deeper understanding of repair pathway regulation, molecular machinery and genome editing outcomes.
Is NHEJ always inaccurate?
No. Classical NHEJ can restore the original sequence, although small insertions or deletions may occur when ends require processing.
Is HDR always error-free?
No. Homology-dependent repair can be high-fidelity, especially when using an appropriate sister chromatid, but template choice, pathway intermediates and context can produce other outcomes.
Are MMEJ and TMEJ identical terms?
They are closely related but not necessarily fully interchangeable. TMEJ refers specifically to polymerase theta-mediated end joining, while MMEJ describes repair using short microhomologies.
Are these the only DNA repair pathways?
No. Other pathways include single-strand annealing and additional homologous recombination mechanisms. Cells also repair many other types of DNA damage.
Non-homologous DNA End Joining
and Alternative Pathways
NATURE REVIEWS MOLECULAR CELL BIOLOGY / 2017
The Molecular Basis and Disease Relevance
of Non-Homologous DNA End Joining
OPEN-ACCESS SCIENTIFIC REVIEW
Error-Prone Repair of DNA
Double-Strand Breaks
REPAIR OUTCOMES AND PATHWAY MECHANISMS
Prime Editing: Mechanistic Insights
and DNA Repair Modulation
REPAIR PATHWAYS IN GENOME ENGINEERING / 2025
DNA Repair — Molecular Biology of the Cell
NCBI BOOKSHELF / FOUNDATIONAL REFERENCE