Homologous Recombination (HR) and the Repair of DNA Double-Strand Breaks
Homologous recombination (HR) is one of two eukaryotic pathways carrying out DNA double-strand break repair (DSB repair). Homologous recombination accurately repairs DNA using the undamaged sister chromatid as a template, maintaining genome stability after introduction of potentially carcinogenic lesions.
Pathway Summary
DNA double-strand breaks (DSB) are genotoxic DNA lesions that pose problems for DNA transcription, replication and segregation. Improper processing of DSB gives rise to chromosomal instability that can result in carcinogenesis through activation of proto-oncogenes or inactivation of tumor suppressor genes. DSB are caused by exogenous sources such as UV radiation, mechanical stress, ionizing radiation, as well as by endogenous sources such as radicals generated during metabolic processes and genotoxic chemicals. Eukaryotes have evolved two distinct pathways of DSB repair- homologous recombination (HR) and non-homologous end joining (NHEJ). The fundamental difference between these pathways is their dependence on DNA homology and accuracy of repair. In general, HR ensures an accurate repair by using the undamaged sister chromatid or homologous chromosome as a template. NHEJ, on the other hand, uses no or extremely limited sequence homology to rejoin ends in a manner that need not be error free.The initial cellular response to DSB is mediated through ATM and the MRN complex (Mre11-Rad50-NBS1). In response to DSB, ATM in effect 'raises the alarm' to DNA damage, phosphorylating many downstream effector targets such as p53, H2AX, Mdm2, BRCA11, c-Abl and Chk2. This swift response acts to halt the cell cycle and stop DNA replication. ATM then facilitates DNA repair or triggers apoptosis based on the severity of the damage. The MRN complex has 3 critical roles in DSB sensing: stabilization, signaling and effector scaffolding. Subsequent steps of DSB repair include DNA end recognition and nucleolytic processing of the broken ends of DNA into 3' end ssDNA. Rad51 along with ssDNA forms the helical nucleoprotein filament that promotes DNA strand exchange. Rad54 interacts with this nucleoprotein filament and stimulates its DNA pairing activity. The binding of RAD54 significantly stabilizes the Rad51 nucleoprotein filament. The Rad54 stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. The roles played by BRCA1 and BRCA2 in DSB repair by HR appear to be somewhat different. Despite the apparent dissimilarity in protein sequence and structure, both BRCA1 and BRCA2 have common biological functions. Both are localized to the nucleus in somatic cells, where they co-exist in characteristic subnuclear foci that redistribute following DNA damage. BRCA2 controls the intracellular transport and function of Rad51. In addition, BRCA2 also appears to control the enzymatic activity of Rad51.Once the homologous DNA has been identified, subsequent steps lead to strand invasion and D-loop formation. The damaged DNA strand invades the undamaged DNA duplex in a process referred to as DNA strand exchange. Upon joint molecule formation and DNA synthesis, branched DNA structures called Holliday junctions can form as late intermediates in HR. Holliday junctions can slide or 'branch-migrate' along the joined DNAs. Branch migration extends the heteroduplex DNA region between identical recombination partners, and might thereby provide a mechanism to prevent recombination between repetitive sequences that are dispersed throughout the genome. A DNA polymerase then extends the 3' end of the invading strand, and subsequent ligation by DNA ligase I yields a heteroduplexed DNA structure. Completion of recombination requires the resolution of Holliday junctions in order to separate the recombining partners. One well-characterized way of resolving Holliday junctions requires the enzymatic action of a resolvase. This recombination intermediate is resolved and the precise, error-free correction of the DSB is complete.
Homologous Recombination (HR) and the Repair of DNA Double-Strand Breaks Genes list
Explore Genes related to Homologous Recombination (HR) and the Repair of DNA Double-Strand Breaks
- ABL proto-oncogene 1, non-receptor tyrosine kinaseABL1
icon_0171_ls_qf_save_program-s - ATM serine/threonine kinaseATM
icon_0171_ls_qf_save_program-s - ATRX chromatin remodelerATRX
icon_0171_ls_qf_save_program-s - BRCA1 DNA repair associatedBRCA1
icon_0171_ls_qf_save_program-s - BRCA2 DNA repair associatedBRCA2
icon_0171_ls_qf_save_program-s - GEN1 Holliday junction 5' flap endonucleaseGEN1
icon_0171_ls_qf_save_program-s - DNA ligase 1LIG1
icon_0171_ls_qf_save_program-s - MRE11 homolog, double strand break repair nucleaseMRE11
icon_0171_ls_qf_save_program-s - nibrinNBN
icon_0171_ls_qf_save_program-s - DNA polymerase alpha 1, catalytic subunitPOLA1
icon_0171_ls_qf_save_program-s - RAD50 double strand break repair proteinRAD50
icon_0171_ls_qf_save_program-s - RAD51 recombinaseRAD51
icon_0171_ls_qf_save_program-s - RAD52 homolog, DNA repair proteinRAD52
icon_0171_ls_qf_save_program-s - replication protein A1RPA1
icon_0171_ls_qf_save_program-s
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What is homologous recombination?
Homologous recombination (HR) repair is a high-fidelity pathway for repairing double-strand breaks (DSBs) in DNA. HR is primarily active during the S and G2 phases of the cell cycle, when a sister chromatid is available as a repair template. This mechanism is especially important for repairing DSBs that occur during DNA replication or in regions of the genome that require precise restoration. HR ensures accurate repair by using a homologous sequence to guide the process, thereby preserving genetic information and minimizing mutations that could lead to genome instability or disease, including cancer.
Homologous recombination steps
The HR DNA repair mechanism is generally broken down into six steps: recognition of the break and resection of the ends, homology search and strand inversion of the sister chromatid, DNA synthesis and extension, second end capture and Holliday junction formation, branch migration and Holliday junction resolution and finally ligation and restoration.
Recognition and resection
Recognizing the presence of a DNA double-strand break is the first step in HR. The MRN complex is the primary sensor, binding to the DNA ends and recruiting the ATM protein kinase, which in turn activates multiple downstream proteins, amplifying the damage signal and recruiting repair proteins. The MRN complex also initiates resection, the process of modifying the exposed ends of the break to form 3’ single-strand DNA overhangs or tails. Extended resection, mediated by two different nucleases, further degrades the 5’ strand extending the 3’ ssDNA tail – a step that is critical for enabling strand invasion. RPA stabilizes and protects the ssDNA ends.
Strand invasion and homology search
In the next step, RPA is replaced by RAD51 which binds to the 3’ ssDNA overhang, forming a nucleoprotein filament. The resulting RAD51-ssDNA filament undertakes a search for the homologous DNA sequence on the sister chromatid, ultimately invading its double-strand DNA once a region of sufficient complementarity is found. The 3’ ssDNA displaces the identical strand, pairing with the complementary strand and forming a D-loop, also known as a displacement loop.
DNA synthesis and extension
As the D-loop structure is held stable, the 3’ ssDNA acts as a primer, initiating DNA synthesis which extends the invading strand beyond the site of the original break using the sister chromatid as a template. When the synthesis-dependent strand annealing (SDSA) pathway is used, only a short DNA segment may be synthesized before the D-loop is disassembled. But when the double Holliday junction pathway is used, DNA synthesis is more extensive.
Second end capture and Holliday junction formation
Once the invading strand is extended, the second resected 3’ overhang from the other side of the break is captured by annealing to the strand that has been displaced on the sister chromatid and extended by DNA synthesis. Together, the result is the formation of two Holliday junctions – four-stranded DNA structures linked by strand exchange.
Branch migration and resolution
Once stabilized by branch migration, the Holliday junctions must be resolved in order to separate the recombined DNA strands. One mechanism of resolution utilizes endonucleases that cut a single strand of DNA from each duplex. This process can produce crossover products where genetic material has been exchanged between the two DNA molecules as well as non-crossover products. A second mechanism utilizes Topoisomerase IIIα to unlink the Holliday junctions, producing only non-crossover products.
Ligation and restoration
After Holliday junction resolution, the final step in the repair process is to seal the single-strand breaks that remain in the DNA backbone, fully restoring integrity of the DNA molecule and restoring the chromatin structure. DNA ligase I is the main enzyme involved in sealing the break, but DNA ligase IIIα can also be involved.
Key proteins involved in homologous recombination
Multiple proteins and complexes are involved in the DNA double-strand break repair process. Key components include the MRN complex, ATM, RAD51, structure-specific endonucleases and the BLM-TopoIIIα-RMI complex.
MRN complex
The MRN complex, a key sensor of double-strand DNA breaks, comprises three proteins: MRE11, RAD50 and NBS1. RAD50 stabilizes the DNA break holding the two strands in close proximity while MRE11, also known as meiotic recombination 11 homolog, is the 3’-5’ exonuclease and endonuclease that initiates resection, trimming the 5’ strands of double-strand breaks to generate the 3’ ssDNA overhangs. NBS1, also known as Nibrin, recruits ATM.
ATM
The serine-threonine kinase ataxia-telangiectasia mutated (ATM) is recruited and bound by the MRN complex. Binding results in a conformational change, leading to ATM activation via autophosphorylation. ATM phosphorylates multiple downstream targets, promoting DNA end resection, recruiting HR DNA repair proteins and inducing cell cycle arrest through checkpoint signaling.
RAD51
After resection of the double-strand break, the resulting 3’ ssDNA overhangs are initially coated by RPA (replication protein A) for protection. RAD51 is then loaded onto the ssDNA, replacing RPA and forming the RAD51-ssDNA nucleoprotein filament. Loading is helped by BRCA2, PALB2 and RAD52.
RAD51-ssDNA nucleoprotein filament scans the sister chromatid for the homologous sequence to use as a template for the repair. Once found, RAD51 promotes strand invasion of the ssDNA into the homologous DNA forming the D-loop where the invading strand pairs with the complementary donor strand. RAD51 is then disassembled, allowing DNA polymerase to extend the annealed strand leading to double Holliday junction (dHJ) formation.
Structure-specific endonucleases
Multiple endonucleases are involved in the resolution of Holliday junctions including GEN1, MUS81-EME1 and SLX1-SLX4. Resolution can result in crossover or non-crossover products.
BLM-TopoIIIα-RMI complex
Comprised of Bloom syndrome protein (BLM), Topoisomerase IIIα and RMI1/2, the BLM-TopoIIIα-RMI complex provides an alternative to Holliday junction resolution. Through the process of dissolution, each strand in the dHJ is reassociated with its original complementary strand resulting in no exchange of genetic information.
Consequences of homologous recombination deficiency
As homologous recombination repair (HRR) primarily operates during the S and G2 phases of the cell cycle and is critical for resolving stalled or collapsed replication forks, deficiency can lead to increased sensitivity to replication stress culminating in chromosomal breaks that are unable to be repaired. Cells with defective homologous recombination put an individual at increased risk of cancer due to accumulation of gross chromosomal rearrangements, which are particularly prominent in BRCA1/2-associated cancers.
Therapeutic relevance of HR in cancer
Cancer cells with homologous recombination (HR) defects are hypersensitive to DNA crosslinking agents and to PARP inhibitors, which block an alternative repair pathway and force accumulation of toxic DSBs. This concept of synthetic lethality has revolutionized targeted cancer therapy and underscores the importance of understanding HR both in health and disease.
References
- Wright WD, Shah SS, Heyer WD. Homologous recombination and the repair of DNA double-strand breaks. J Biol Chem. 2018 ;293(27):10524–10535.
- Ceccaldi R, Rondinelli B, D'Andrea AD. Repair pathway choices and consequences at the double-strand break. Trends Cell Biol. 2016;26(1):52–64.