DNA double-strand breaks (DSB) are considered the most lethal form of DNA damage which can be induced by a number of different endogenous and exogenous factors. Since unrepaired or misrepaired DSB can initiate processes leading to mutagenesis, tumorigenesis and cell death, repair of DSB is essential to maintain genome stability and cell viability. Cells have evolved two major pathways for the repair of DSB, homologous recombination (HR) and non-homologous end-joining (NHEJ).

  

 

 

 

 

 

 

 

 

 

 

 

 


 

 

 

 

 

Models for DSB repair. Homologous recombination. In this model, red and blue double-stranded DNA (dsDNA) represent homologous sequences. At the DSB site, dsDNA (shown in red) is exonucleolytically processed to form 3' single-stranded DNA (ssDNA) tails, which invade homologous intact sequences (shown in blue). DNA strand exchange follows and generates a joint molecule between damaged and undamaged duplex DNAs. Sequence information that is missing at the DSB site is restored by DNA synthesis (resynthesized DNA is shown in green). The interlinked molecules are then processed by branch migration (indicated by right and left arrows), Holliday junction resolution and DNA ligation. Non-homologous end-joining. Following DSB formation, broken DNA ends are processed to yield appropriate substrates for direct ligation. No homology is necessary for DSB repair by NHEJ. Breaks can be joined accurately, but more often, small insertions or deletions are created [Adapted from Dudáš and Chovanec (2004) Reviews in Mutation Research 566:131-167].

 

Homologous recombination

 

For many years, HR had been considered to be a minor pathway that acts on DSB in higher eukaryotes. It was therefore believed that DSB repair in these organisms is almost exclusively undertaken by NHEJ (see below). Later, however, it was established that HR also contributes considerably to DSB repair in higher eukaryotes. In Saccharomyces cerevisiae, HR is the favored DSB repair pathway, whereas NHEJ is only of minor importance. Basically, in S. cerevisiae NHEJ activity can be demonstrated only in the absence of HR and it may thus serve only as a backup system. The HR components in this organism belong to the RAD52 epistasis group, which includes RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11 and XRS2 genes. Cells mutated in these genes are in general sensitive to ionizing radiation, unable to repair DSB, and defective in mitotic and/or meiotic recombination.

 

The RAD52 epistasis group HR factors in yeast and human

 

Yeast

Human

Biochemical activity and function

Rad50

RAD50

ATP-dependent DNA binding activity; ATPase activity

Mre11

MRE11

3' → 5' dsDNA exonuclease and ssDNA endonuclease activities; DNA binding and ssDNA annealing activities; DNA duplex unwinding and hairpin cleavage activities

Xrs2

NBS1

Human: regulates DNA duplex unwinding and nuclease activities of the MRE11-RAD50-NBS1 complex; recruits the complex to vicinity of DNA damage 

Rad51

RAD51

ATP-dependent DNA binding, homologous pairing, and DNA strand exchange activities

-

RAD51B

DNA binding, protein kinase, and DNA-stimulated ATPase activities

-

RAD51C

DNA binding and DNA-stimulated ATPase activities; ATP-independent DNA strand exchange activity; ATP-independent and Mg2+-dependent homologous pairing activity

-

RAD51D

DNA binding and DNA-stimulated ATPase activities; ATP-independent and Mg2+-dependent homologous pairing activity; protects telomeres against attrition and fusion

Rad52

RAD52

DNA binding, ssDNA annealing, weak DNA strand exchange, and weak homologous pairing activities

Rad54

RAD54

DNA binding, dsDNA-dependent ATPase, dsDNA unwinding, dsDNA topology-modifying, chromatin remodeling, and branch migration activities

Rad55

XRCC2

Yeast: ATPase activity; serves as a terminal target of the DNA damage checkpoints coordinating the cellular response to genotoxic stress; Human: DNA binding and ATP-independent and Mg2+-dependent homologous pairing activities

Rad57

XRCC3

Yeast: ATPase activity; Human: DNA binding and ATP-independent and Mg2+-dependent homologous pairing activities

Rad59

-

DNA binding and ssDNA annealing activities

 

DNA non-homologous end-joining

 

NHEJ process was initially discovered in mammalian cells, where it represents the main DSB repair pathway, and where the core NHEJ factors are the DNA-dependent protein kinase (DNA-PK), consisting of the catalytic subunit (DNA-PKcs) and the two DNA end-binding components (KU70 and KU80), the DNA ligase IV-XRCC4 complex associating with recently identified CERNUNNOS/XLF protein, and the ARTEMIS protein. NHEJ has also been discovered in other organisms including the budding yeast S. cerevisiae, where the KU70, KU80, DNA ligase IV and XRCC4 homologues have been identified (YKu70, YKu80, Dnl4 and Lif1, respectively). Although S. cerevisiae lacks clear homologues of DNA-PKcs and ARTEMIS, it is endowed with an additional regulatory NHEJ factor, Nej1, which appears to be genuine homologue of CERNUNNOS/XLF.

 

NHEJ factors in yeast and human

Yeast

Human

Biochemical activity/function

Yku70-Yku80

KU70-KU80

DNA ends-binding complex; DNA-dependent ATPase; ATP-dependent DNA helicase; protects DNA ends from degradation; recruits DNA-PKcs when bound to DNA ends; recruits DNA ligase IV-XRCC4 (Dnl4-Lif1) to site of DSB; stimulates DNA ligase IV-XRCC4 (Dnl4-Lif1)-mediated ligation

-

DNA-PKcs

ATP-dependent serine/threonine protein kinase activity; binds to DNA ends as well as to DNA ends-bound KU70-KU80; synapsis and regulatory factor in NHEJ; phosphorylates the downstream targets

-

ARTEMIS

5' → 3' exonuclease activity; upon complex formation with, and subsequent phosphorylation by, DNA-PKcs it acquires also endonucleolytic activity on 5' and 3' overhangs, as well as hairpins; involved in nucleolytic end-processing step of NHEJ

Mre11-Rad50-Xrs2

MRE11-RAD50-NBS1

For details on Mre11-Rad50-Xrs2, see Table above; the human complex is presumably not involved in NHEJ

Dnl4-Lif1

DNA ligase IV-XRCC4

Complex binds to DNA; XRCC4 (Lif1) modulates stability and activity of DNA ligase IV (Dnl4) and targets the enzyme to the site of DSB; DNA ligase IV (Dnl4) is an ATP-dependent dsDNA ligase

Nej1

CERNUNNOS/XLF

CERNUNNOS/XLF binds to DNA; stimulates the ligase activity of DNA ligase IV-XRCC4 through a mechanism that involves specific, direct action with this complex

Pol4

DNA polymerase μ or λ

DNA synthesis activity; functions in the gap-filling step in NHEJ

Rad27

FEN-1

Structure-specific nuclease that possesses flap endonuclease and 5' → 3' exonuclease activities; acts at a subset of NHEJ events