What is Dr. Zubair Khalid's research focus?

Dr. Zubair Khalid specializes in molecular virology, mRNA vaccine development, and computational biology, with a focus on avian pathogens like IBDV and Avian Reovirus.

Where is Dr. Zubair Khalid currently working?

Dr. Zubair Khalid is a Postdoctoral Research Associate at the University of Maryland (UMD), specifically within the Department of Animal and Avian Sciences.

Non-Homologous End Joining Proteins: Mechanism and Repair

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to Non-Homologous End Joining (NHEJ)

DNA double-strand breaks (DSBs) are among the most cytotoxic forms of DNA damage. A single unrepaired DSB can trigger cell cycle arrest, senescence, or apoptosis, and misrepair can generate chromosomal translocations that drive oncogenesis. DSBs arise from exogenous sources such as ionizing radiation and chemotherapeutic agents, and from endogenous events including replication fork collapse, oxidative stress, and programmed genomic rearrangements during V(D)J recombination in developing lymphocytes.

Cells have evolved two principal pathways to repair DSBs: non-homologous end joining (NHEJ) and homologous recombination (HR). This article focuses on the proteins that execute NHEJ, the dominant DSB repair pathway in mammalian cells.

Overview of DNA Double-Strand Breaks

A DSB is defined as a break in both strands of the DNA duplex with broken ends that can be physically separated. Unlike single-strand breaks, which retain a complementary template for repair synthesis, DSBs lack an intact sister chromatid or homologous chromosome in close proximity during most of the cell cycle. This creates a unique challenge: the repair machinery must either find a homologous template (HR) or directly rejoin the broken ends (NHEJ).

NHEJ operates throughout the cell cycle but predominates in G0/G1 phase, when sister chromatids are unavailable. It is a template-independent process that directly ligates broken DNA ends. The pathway is often described as "error-prone" because it can introduce small insertions or deletions (indels) at the repair junction, but this characterization requires nuance—many NHEJ events are precise, particularly when the broken ends are compatible.

NHEJ vs. Homologous Recombination

The fundamental distinction between NHEJ and HR lies in template usage. HR requires a homologous DNA sequence—typically the sister chromatid—to serve as a template for accurate repair. This restricts HR to the S and G2 phases of the cell cycle, when sister chromatids exist. HR is essentially error-free.

NHEJ, by contrast, requires no homology. The Ku70/Ku80 heterodimer binds directly to DNA ends, and the repair machinery processes and ligates them without reference to a template. This simplicity allows NHEJ to function in any cell cycle phase, but it comes at the cost of potential sequence alterations at the junction. For a detailed comparison of the two pathways, see Homologous Recombination.

The choice between NHEJ and HR is not random; it is regulated by cell cycle phase, chromatin context, and the activity of key regulatory proteins discussed in Section 4.

Core NHEJ Proteins and Their Functions

The core NHEJ machinery comprises seven proteins that assemble sequentially at the break site. Each plays a defined role in recognizing, processing, or ligating DNA ends.

Ku70/Ku80 Heterodimer

The Ku heterodimer, composed of Ku70 (XRCC6, 70 kDa) and Ku80 (XRCC5, 86 kDa), is the first protein to bind DSB ends. Ku is extraordinarily abundant—approximately 400,000 molecules per human cell—and has a remarkable affinity for DNA ends, with a dissociation constant (Kd) in the picomolar range.

Structurally, Ku70 and Ku80 form a basket-shaped heterodimer that threads onto the DNA double helix like a ring. The protein makes extensive contacts with the sugar-phosphate backbone over approximately 20 base pairs, but makes no sequence-specific contacts. This explains Ku's ability to bind any DNA end regardless of sequence.

Once bound, Ku serves multiple functions. It protects the DNA ends from exonuclease degradation, recruits downstream NHEJ factors, and translocates inward along the DNA in an ATP-independent manner, allowing multiple Ku molecules to load onto a single DNA molecule. Ku also has weak ATPase and helicase activities, though their physiological significance remains debated.

DNA-Dependent Protein Kinase Catalytic Subunit

DNA-PKcs (encoded by PRKDC, ~470 kDa) is the largest subunit of the DNA-dependent protein kinase (DNA-PK) holoenzyme. DNA-PKcs is recruited to DNA ends by Ku, and together they form the active DNA-PK complex. DNA-PKcs is a serine/threonine kinase belonging to the phosphatidylinositol 3-kinase-related kinase (PIKK) family, which also includes ATM and ATR.

The kinase activity of DNA-PKcs is activated only when it is bound to DNA ends in complex with Ku. Once activated, DNA-PKcs autophosphorylates at multiple sites, including the ABCDE cluster (Ser2612, Ser2624, Ser2638, Thr2639, Ser2640) and the PQR cluster (Ser2023, Ser2029, Ser2035, Ser2036, Ser2041, Ser2051, Ser2052, Ser2053, Ser2056). Autophosphorylation induces a conformational change that opens the DNA-PKcs molecule, allowing access of processing enzymes to the DNA ends.

DNA-PKcs also phosphorylates downstream substrates, including Artemis, XRCC4, XLF, and numerous other proteins. The kinase activity is essential for NHEJ; cells expressing kinase-dead DNA-PKcs are severely radiosensitive and defective in DSB repair.

Artemis Nuclease

Artemis (encoded by DCLRE1C) is a structure-specific nuclease that processes DNA ends before ligation. It exists in a stable complex with DNA-PKcs and is activated when DNA-PKcs phosphorylates it. Artemis has two distinct nuclease activities: an intrinsic 5'→3' exonuclease activity and an endonuclease activity that opens DNA hairpins.

The endonuclease activity is critical for V(D)J recombination, where it opens the hairpin intermediates generated by RAG proteins at coding joints. The exonuclease activity is required for processing overhanging ends during NHEJ. For example, if a DSB has a 3' overhang that cannot be ligated directly, Artemis can trim it to create blunt or compatible ends.

Artemis is not required for all NHEJ events. Simple DSBs with compatible blunt ends can be ligated without Artemis. However, when ends are damaged or incompatible—as occurs after ionizing radiation—Artemis is essential for processing.

Ligation Complex: XRCC4, DNA Ligase IV, XLF

The final step of NHEJ is DNA ligation, performed by DNA Ligase IV (encoded by LIG4) in complex with XRCC4 (X-ray repair cross-complementing protein 4, encoded by XRCC4) and XLF (XRCC4-like factor, also called Cernunnos, encoded by NHEJ1).

DNA Ligase IV is a 96 kDa protein that catalyzes phosphodiester bond formation between adjacent 3'-hydroxyl and 5'-phosphate termini. Unlike other DNA ligases, DNA Ligase IV can ligate ends with short gaps or incompatible overhangs, though with reduced efficiency. It is also uniquely able to ligate ends that are not perfectly base-paired, a property that contributes to NHEJ's flexibility.

XRCC4 serves as a scaffold and stabilizer for DNA Ligase IV. The XRCC4-Ligase IV complex is stable and abundant in cells. XRCC4 also interacts with other NHEJ factors and with chromatin, helping to position the ligase at the break site.

XLF is structurally similar to XRCC4 and forms filaments with it. The XRCC4-XLF filament can bridge DNA ends, promoting their alignment and stimulating ligation by DNA Ligase IV. This filament formation is particularly important for ligating non-compatible ends that require synapsis across a gap.

The following table summarizes the core NHEJ proteins and their primary functions:

Protein	Gene	Size	Primary Function
Ku70	XRCC6	70 kDa	DNA end binding, recruitment of downstream factors
Ku80	XRCC5	86 kDa	DNA end binding, stabilization of Ku70
DNA-PKcs	PRKDC	470 kDa	Kinase activity, end processing coordination
Artemis	DCLRE1C	78 kDa	Endonucleolytic hairpin opening, 5'→3' exonuclease
XRCC4	XRCC4	38 kDa	Scaffold for Ligase IV, DNA end bridging
DNA Ligase IV	LIG4	96 kDa	Catalyzes phosphodiester bond formation
XLF	NHEJ1	33 kDa	Filament formation, end alignment, ligation stimulation

Step-by-Step Mechanism of NHEJ

The NHEJ reaction proceeds through three phases: recognition and tethering, end processing, and ligation. These phases are temporally ordered but not strictly sequential—processing and ligation can occur iteratively if initial ligation attempts fail.

Recognition and Tethering of DNA Ends

Ku loading: The Ku70/Ku80 heterodimer binds to each broken DNA end within seconds of break formation. Ku has no sequence specificity and binds with high affinity to any free DNA end. The binding is essentially irreversible under physiological conditions.

DNA-PKcs recruitment: Ku-bound DNA ends recruit DNA-PKcs. The Ku-DNA complex has a higher affinity for DNA-PKcs than free DNA, ensuring that DNA-PKcs is recruited specifically to Ku-bound ends. Each DNA end now has a Ku heterodimer and a DNA-PKcs molecule, forming the DNA-PK holoenzyme.

Synapsis: The two DNA-PK complexes at opposite ends of the break interact with each other, bringing the DNA ends into close proximity. This synaptic complex aligns the ends for processing and ligation. The interaction between DNA-PKcs molecules is mediated by the C-terminal region of the protein and is enhanced by autophosphorylation.

Kinase activation: DNA-PKcs autophosphorylates in trans—each molecule phosphorylates the other within the synaptic complex. This autophosphorylation causes a conformational rearrangement that opens the DNA-PKcs structure, exposing the DNA ends to processing enzymes.

End Processing and Trimming

Once the synaptic complex is formed and DNA-PKcs has autophosphorylated, the DNA ends must be made compatible for ligation. This processing step is the most variable part of NHEJ and depends entirely on the nature of the break.

Compatible blunt ends: If the DSB has blunt ends with 5'-phosphate and 3'-hydroxyl groups, no processing is required. The ends can proceed directly to ligation.

Compatible overhangs: If the DSB has complementary 5' or 3' overhangs (e.g., a 4-base 5' overhang with complementary sequence on the opposite end), the overhangs can anneal and the remaining nicks can be ligated. This is a precise repair event that does not alter the sequence.

Incompatible overhangs: If the overhangs are non-complementary, they must be processed. Artemis, activated by DNA-PKcs phosphorylation, can trim 5' and 3' overhangs. The nuclease activity is structure-specific—it cleaves at single-strand/double-strand transitions and can open hairpins.

Damaged ends: Ionizing radiation frequently produces ends with damaged nucleotides, abasic sites, or sugar modifications. These must be removed before ligation. The polynucleotide kinase phosphatase (PNKP) removes 3'-phosphate groups and adds 5'-phosphate groups. Aprataxin (APTX) removes adenylate groups from abortive ligation intermediates. Other enzymes, including DNA polymerases μ and λ, can fill in gaps to create compatible ends.

The processing step is where NHEJ can introduce sequence alterations. Trimming of overhangs removes nucleotides, and gap filling by polymerases adds nucleotides. The net result is often a small deletion or insertion at the junction.

Ligation and Repair Completion

Ligase recruitment: The XRCC4-DNA Ligase IV complex is recruited to the break, likely through interactions with Ku and DNA-PKcs. XLF is also recruited and forms filaments with XRCC4 that bridge the two DNA ends.

Adenylation: DNA Ligase IV first reacts with ATP to form a covalent ligase-AMP intermediate. The AMP is transferred to the 5'-phosphate at the DNA end, activating it for nucleophilic attack by the 3'-hydroxyl of the opposite end.

Phosphodiester bond formation: The 3'-hydroxyl attacks the activated 5'-phosphate, forming a phosphodiester bond and releasing AMP. This seals the nick and completes the repair.

Disassembly: After ligation, the NHEJ proteins must be removed from the DNA. Ku is removed by a process involving ubiquitylation and proteasomal degradation, though the exact mechanism remains under investigation. DNA-PKcs dissociates after autophosphorylation. The repair is complete when the DNA is restored to its original duplex state.

The entire NHEJ reaction can occur in minutes for simple breaks. Complex breaks with damaged ends may require hours and multiple rounds of processing and attempted ligation.

Regulation of NHEJ Choice

The decision to repair a DSB by NHEJ versus HR has profound consequences for genome stability. This choice is regulated at multiple levels, with cell cycle phase being the dominant determinant.

Cell Cycle Control

HR requires a sister chromatid as a template and is therefore restricted to S and G2 phases. NHEJ can operate in any phase but is particularly important in G0/G1, when HR is impossible.

The cyclin-dependent kinase (CDK) system controls this choice. CDK activity is low in G1 and high in S/G2. CDK phosphorylation of key HR proteins, including CtIP and BRCA2, promotes HR in S/G2. In G1, the absence of CDK activity prevents HR initiation, and DSBs are channeled into NHEJ.

Competition with Homologous Recombination

At the molecular level, the choice between NHEJ and HR is governed by a competition between 53BP1 and BRCA1 at the break site.

53BP1 (p53-binding protein 1) is recruited to DSBs in G1 phase. It promotes NHEJ by recruiting downstream effectors, including RIF1 and the shieldin complex, which block resection of DNA ends. Resection—the 5'→3' degradation of DNA ends to create 3' single-stranded overhangs—is the first committed step of HR. By blocking resection, 53BP1 forces repair into the NHEJ pathway.

BRCA1 is recruited to DSBs in S/G2 phase. It counteracts 53BP1 and promotes resection, committing the break to HR. BRCA1 also recruits additional resection factors, including CtIP and the MRN complex (MRE11-RAD50-NBS1).

The antagonism between 53BP1 and BRCA1 is a central regulatory node. In cells lacking BRCA1, DSBs in S/G2 are inappropriately repaired by NHEJ, leading to genomic instability and cancer predisposition. Conversely, loss of 53BP1 can restore HR in BRCA1-deficient cells, a finding with therapeutic implications for PARP inhibitor sensitivity.

Additional regulatory inputs include the ATM kinase, which phosphorylates both 53BP1 and BRCA1, and the chromatin context at the break site. For a broader view of the repair pathway landscape, see Non Homologous End Joining Pathway.

Methods to Study NHEJ Proteins

Investigating NHEJ requires experimental systems that can detect and quantify repair events. Several complementary approaches are commonly used.

In Vitro End-Joining Assays

Biochemical assays reconstitute NHEJ using purified proteins or cell extracts. A typical reaction contains:

Linearized plasmid DNA with defined ends (blunt, 5' overhang, or 3' overhang)
Cell extract (or purified Ku, DNA-PKcs, Artemis, XRCC4, Ligase IV, XLF)
Reaction buffer: 25 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl₂, 1 mM ATP, 1 mM DTT
Incubation at 37°C for 30–60 minutes

Products are analyzed by agarose gel electrophoresis. Successful ligation produces higher-molecular-weight products (dimers, concatemers) from monomeric substrates. The efficiency and fidelity of ligation can be quantified by transforming ligated products into bacteria and sequencing individual clones.

This approach has defined the minimal protein requirements for NHEJ and has revealed the biochemical activities of individual factors. For example, such assays demonstrated that Ku, DNA-PKcs, XRCC4, and Ligase IV are sufficient for ligation of compatible ends, while Artemis is required for processing of incompatible ends.

GFP-Based Reporter Systems

Cell-based reporter assays measure NHEJ efficiency in living cells. The most widely used system is the GFP reporter integrated into a chromosomal locus.

The reporter contains a GFP coding sequence interrupted by an endonuclease recognition site (e.g., I-SceI or Cas9). The recognition site is flanked by a stop codon and a downstream GFP fragment. When the endonuclease is expressed, it creates a DSB. If NHEJ repairs the break and removes the stop codon, GFP is expressed and cells become fluorescent.

The frequency of GFP-positive cells, measured by flow cytometry, reflects NHEJ efficiency. This system can be adapted to measure mutagenic versus precise NHEJ by sequencing the repair junctions from individual GFP-positive clones. For applications in genome editing, see Non Homologous End Joining CRISPR.

Live-Cell Imaging of Repair Foci

Fluorescence microscopy allows visualization of NHEJ protein recruitment to DSBs in real time. The standard approach uses laser microirradiation to create localized DSBs in a defined nuclear region, followed by time-lapse imaging of fluorescently tagged NHEJ proteins.

Ku-GFP and DNA-PKcs-GFP accumulate at laser-induced damage within seconds. XRCC4-GFP and Ligase IV-GFP arrive later, consistent with their roles in the ligation step. Fluorescence recovery after photobleaching (FRAP) can measure protein mobility and binding kinetics.

Immunofluorescence of fixed cells is used to detect endogenous repair foci. γ-H2AX (phosphorylated histone H2AX) marks DSBs, and co-staining with antibodies against 53BP1, BRCA1, or other repair factors reveals pathway choice. For protein identification in complex mixtures, Mass Spectrometry Work for Proteins and Mass Spec Identify Proteins provide complementary approaches to define the NHEJ interactome.

Clinical Significance and Disease Links

Mutations in NHEJ genes cause human diseases characterized by immunodeficiency, radiosensitivity, and cancer predisposition. The clinical phenotypes reflect the dual roles of NHEJ in DSB repair and V(D)J recombination.

NHEJ Deficiencies and Immunodeficiency

V(D)J recombination is the process by which developing lymphocytes assemble antigen receptor genes from variable (V), diversity (D), and joining (J) gene segments. This process generates DSBs as intermediates and requires NHEJ for resolution. Consequently, defects in NHEJ proteins cause severe combined immunodeficiency (SCID).

Artemis deficiency (caused by DCLRE1C mutations) is the most common NHEJ-related SCID. Patients have no T cells or B cells (T-B-NK+ SCID) because V(D)J recombination cannot complete. They also exhibit profound radiosensitivity.

DNA Ligase IV deficiency (LIG4 mutations) causes a milder immunodeficiency with additional features including microcephaly, developmental delay, and bone marrow failure. The phenotype is more severe than Artemis deficiency because Ligase IV is required for all NHEJ, not just V(D)J recombination.

XLF deficiency (NHEJ1 mutations) causes a SCID-like phenotype with growth retardation. The immunodeficiency is often less severe than Artemis deficiency, suggesting partial functional redundancy.

DNA-PKcs deficiency (PRKDC mutations) is rare in humans but well-characterized in animal models. The equine and canine forms of SCID are caused by DNA-PKcs mutations.

NHEJ in Cancer and Radiosensitivity

NHEJ defects are associated with cancer predisposition, particularly lymphoid malignancies. The chromosomal translocations that characterize many leukemias and lymphomas arise from aberrant V(D)J recombination or off-target NHEJ events.

Heterozygous mutations in NHEJ genes are found in some cancer families, and somatic mutations are common in tumors. However, complete loss of NHEJ is often cell-lethal or causes severe genomic instability, so tumors typically retain at least one functional NHEJ allele.

NHEJ status is also a determinant of radiosensitivity. Cells with defective NHEJ are exquisitely sensitive to ionizing radiation because they cannot repair radiation-induced DSBs. This has therapeutic implications: tumors with NHEJ defects may be more responsive to radiation therapy, and NHEJ inhibitors are being developed as radiosensitizers.

Conversely, overexpression of NHEJ proteins in tumors can confer radioresistance. For example, DNA-PKcs is overexpressed in many cancer types, and its expression correlates with poor response to radiotherapy.

Common Pitfalls and Misconceptions

Students frequently misunderstand several aspects of NHEJ. The following clarifications address the most common errors.

NHEJ is Error-Prone but Not Always Mutagenic

The characterization of NHEJ as "error-prone" leads students to assume that every NHEJ event introduces mutations. This is incorrect. When DSB ends are compatible—blunt ends or complementary overhangs—NHEJ ligates them precisely with no sequence alteration. Mutations arise only when ends require processing, and even then, the alterations are typically small (1–10 base pairs).

The distinction matters clinically. Precise NHEJ is essential for V(D)J recombination, where junctional diversity is generated by programmed processing, not by random error. It is also important for genome editing, where NHEJ is used to create targeted knockouts—the indels are the desired outcome, not an unavoidable byproduct.

DNA-PKcs is Not the Only Kinase

Students sometimes assume that DNA-PKcs is the sole kinase involved in NHEJ. In fact, ATM (ataxia-telangiectasia mutated) also phosphorylates NHEJ factors and regulates the pathway. ATM is activated by DSBs and phosphorylates 53BP1, promoting its recruitment to break sites. ATM also phosphorylates Artemis and DNA-PKcs themselves.

The two kinases have distinct but overlapping functions. DNA-PKcs is essential for NHEJ catalysis, while ATM is more important for checkpoint activation and pathway choice. Cells lacking ATM are radiosensitive but not completely NHEJ-deficient, whereas cells lacking DNA-PKcs are severely NHEJ-defective.

Ku is Not a Simple "End-Capping" Protein

Ku is often described as a "cap" that protects DNA ends, implying a passive role. In reality, Ku is an active scaffold that recruits every downstream NHEJ factor. It also translocates along DNA, can slide past nucleosomes, and participates in telomere maintenance and transcriptional regulation.

Moreover, Ku is not always beneficial. At dysfunctional telomeres or during replication, Ku binding can interfere with other processes. Cells have evolved mechanisms to remove Ku from DNA, including ubiquitylation and the action of the p97/VCP segregase.

NHEJ and HR Are Not Mutually Exclusive

The textbook view of NHEJ and HR as competing pathways suggests that a given DSB is repaired by one or the other. In reality, the pathways can cooperate. If NHEJ fails to ligate ends, resection can initiate and the break can be shunted into HR. Conversely, HR intermediates can be resolved by NHEJ-like ligation.

The 53BP1-BRCA1 antagonism is better understood as a dynamic balance than a binary switch. The outcome depends on the duration of resection, the availability of HR factors, and the cell cycle phase.

Summary and Study Tips

Key Takeaways

NHEJ is the dominant DSB repair pathway in mammalian cells, operating throughout the cell cycle but especially in G0/G1.
The core NHEJ machinery comprises Ku70/Ku80, DNA-PKcs, Artemis, XRCC4, DNA Ligase IV, and XLF.
Ku binds DNA ends within seconds and recruits the remaining factors in a defined order.
DNA-PKcs is a kinase that autophosphorylates and phosphorylates downstream substrates, including Artemis.
Artemis processes incompatible ends; XRCC4-Ligase IV-XLF performs the ligation.
NHEJ can be precise or mutagenic, depending on the nature of the DNA ends.
The 53BP1-BRCA1 axis controls NHEJ versus HR choice.
NHEJ defects cause SCID, radiosensitivity, and cancer predisposition.

Mnemonics and Memory Aids

Order of protein recruitment: "Ku Picks Artemis, X-ray Ligase X" — Ku, DNA-PKcs, Artemis, XRCC4, Ligase IV, XLF.

Ku structure: "Ku is a donut" — it threads onto DNA like a ring.

DNA-PKcs kinase: "PKcs is the big one" — 470 kDa, the largest NHEJ protein.

53BP1 vs BRCA1: "53BP1 Blocks resection, BRCA1 Begins resection" — both start with B, but they have opposite effects.

NHEJ vs HR: "NHEJ is No Homology, HR is Homology Required" — the names say it all.

For exam preparation, focus on the order of protein recruitment, the specific functions of each protein, and the regulatory logic of pathway choice. Practice drawing the pathway from memory, including the processing steps for different types of ends.

Frequently Asked Questions

What are non-homologous end joining proteins?

Non-homologous end joining proteins are the seven core factors—Ku70, Ku80, DNA-PKcs, Artemis, XRCC4, DNA Ligase IV, and XLF—that recognize, process, and ligate DNA double-strand breaks without requiring sequence homology. They function as a coordinated machine that binds broken DNA ends, brings them together, and seals the break.

How does NHEJ differ from homologous recombination?

NHEJ directly ligates broken DNA ends without a template, while homologous recombination uses a sister chromatid as a template for accurate repair. NHEJ operates in all cell cycle phases but predominates in G0/G1; HR is restricted to S/G2. NHEJ can introduce small indels at the junction, while HR is essentially error-free.

What is the role of Ku70/Ku80 in NHEJ?

Ku70/Ku80 is the DNA end-binding heterodimer that initiates NHEJ. It binds to broken DNA ends with high affinity, protects them from degradation, and recruits DNA-PKcs and other downstream factors. Ku is the molecular scaffold on which the entire NHEJ reaction is built.

Why is NHEJ considered error-prone?

NHEJ is considered error-prone because end processing can remove or add nucleotides at the junction. Artemis trimming of overhangs deletes bases, and DNA polymerases μ and λ can add bases during gap filling. However, NHEJ is precise when ends are compatible, and the error rate is much lower than often portrayed.

What happens if NHEJ is defective?

NHEJ defects cause severe combined immunodeficiency (SCID) because V(D)J recombination requires NHEJ. Patients also exhibit profound radiosensitivity, growth defects, and increased cancer risk, particularly for lymphoid malignancies. The severity depends on which protein is affected.

How is NHEJ regulated during the cell cycle?

NHEJ is available throughout the cell cycle but is the primary pathway in G0/G1, when sister chromatids are absent. CDK activity in S/G2 promotes HR by activating resection factors. The 53BP1 protein blocks resection in G1, channeling breaks into NHEJ, while BRCA1 counteracts 53BP1 in S/G2 to promote HR.

What is the function of DNA-PKcs in NHEJ?

DNA-PKcs is the catalytic subunit of the DNA-PK holoenzyme. It is a serine/threonine kinase that autophosphorylates upon DNA binding, inducing conformational changes that expose DNA ends for processing. DNA-PKcs also phosphorylates Artemis, activating its nuclease activity, and phosphorylates other NHEJ factors to coordinate the repair reaction.