Pathway choice in DNA double strand break repair: observations of a balancing act

Abstract

Proper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment.

Figure 1

HR and NHEJ. NHEJ) NHEJ starts with recognition of the DNA ends by the Ku70/80 heterodimer, which recruits DNA-PKcs. If the ends are incompatible, nucleases such as Artemis can trim the ends. The XRCC4-DNA Ligase IV-XLF ligation complex seals the break. HR) The MRN-CtIP-complex starts resection on the breaks to generate single stranded DNA (ssDNA). After resection the break can no longer be repaired by NHEJ. The ssDNA is first coated by RPA, which is subsequently replaced by Rad51 with the help of BRCA2. These Rad51 nucleoprotein filaments mediate strand invasion on the homologous template. Extension of the D-loop and capture of the second end lead to repair.

Figure 2

NHEJ repair assays. a) Linear plasmid DNA with 6 bp repeats at the ends is joined after transfection. The joints are amplified by PCR and digested using BstXI to distinguish between direct repair and microhomology mediated repair [21]. b) Repair of linearized plasmid DNA results in restoration of GFP expression. c) Cleavage by I-SceI and subsequent repair lead to loss of the middle splice donor and acceptor sites (SD and SA) and the adenoviral exon (AD), resulting in the expression of active GFP [22]. d) H2Kd fused to CD8 is expressed from the intact substrate. Repair of the oppositely oriented I-SceI breaks results in loss of H2Kd-CD8 and allows expression of CD4 [23]. e) Similar to d), the intact substrate expresses GFP, while the repaired substrate allows expression of RFP and loses GFP expression [24]. f) Between the opposite I-SceI sites, a translation start site is located, preventing translation of the XHATM resistance gene. Repair of the I-SceI breaks and loss of the intervening ATG results in XHATM resistance. The sequence around the breaks can be sequenced to monitor loss of nucleotides [25]. g) V(D)J recombination assay. Cleavage by the Rag1/2 endonuclease at the recombination signal sequences induces inversion of the intervening sequence. Small arrows indicate location of PCR primers to amplify joints [21].

Figure 3

HR Assay. The 5’ GFP is inactivated by several in frame stop codons and contains an I-SceI site. A downstream truncated GFP, lacking the I-SceI sites and stops, serves as a template. Accurate repair via HR results in GFP expression.

Figure 4

BRCA1 and 53BP1 in DSB repair. a) Repair of replication associated breaks requires HR. 53BP1 blocks resection of the one-ended break in BRCA1 deficient cells, preventing repair via HR. The breaks are either left unrepaired or repaired via NHEJ using other random DNA ends, which leads to chromosomal rearrangements and genomic instability. In the absence of 53BP1, resection of the DNA ends can take place, allowing faithful repair via HR. b) IR induced two ended DSBs are mainly repaired via NHEJ, however part of the breaks is repaired via HR or alternative end-joining (alt-EJ). Repair via HR or alt-EJ increases when classical NHEJ is impaired by a mutation in one of the core NHEJ genes or 53BP1.