Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells

Abstract

Chromosomal double-strand breaks (DSBs) in mammalian cells are repaired by either homology-directed repair (HDR), using a homologous sequence as a repair template, or nonhomologous end-joining (NHEJ), which often involves sequence alterations at the DSB site. To characterize the interrelationship of these two pathways, we analyzed HDR of a DSB in cells deficient for NHEJ components. We find that the HDR frequency is enhanced in Ku70(-/-), XRCC4(-/-), and DNA-PKcs(-/-) cells, with the increase being particularly striking in Ku70(-/-) cells. Neither sister-chromatid exchange nor gene-targeting frequencies show a dependence on these NHEJ proteins. A Ku-modulated two-ended versus one-ended chromosome break model is presented to explain these results.

Figure 1

HDR is elevated in NHEJ mutant cell lines. (A) The hprtDRGFP targeting vector contains homologous hprt targeting arms, for integration of the HDR reporter substrate DR-GFP into the hprt locus, and a dominant selectable puro^R marker. In the DR-GFP substrate, the iGFP gene has 0.8 kb of sequence homology to direct repair of an I-SceI-cleaved SceGFP gene, to restore functionality to the GFP gene. (B) Flow cytometric analysis of ES cells containing the targeted hprtDRGFP reporter after electroporation with an I-SceI expression vector. The GFP-positive population is shifted “greenward” from the GFP-negative population. (DPKcs) DNA-PKcs. (C) Summary of the percentage of GFP-positive cells from each of the electroporated cell lines. Bars represent the average of three independently isolated hprtDRGFP subclones for each cell line. Error bars are ±1 S.D. (N = 3).

Figure 2

NHEJ mutants show increased I-SceI site loss after DSB repair. (A) PCR assay to detect DSB repair. The genomic region surrounding the I-SceI break site was amplified from cell lines transfected with the I-SceI expression vector using primers shown in Figure 1A. PCR products were digested with I-SceI and resolved on agarose gels. (B) Fraction of I-SceI site loss from transfected cell populations. I-SceI⁺ and I-SceI⁻ PCR products were quantified, with the fraction of amplified product from each cell population no longer digestible by I-SceI indicated. Note that because the smaller band incorporates proportionately less ethidium bromide, band intensities were normalized for product length. Error bars are ±1 S.D. (N = 3). (C) Fraction of I-SceI⁻ PCR product from each population derived from HDR. The fraction of GFP-positive cells was normalized to the fraction of I-SceI⁻ PCR product from each transfected cell population. Error bars are ±1 S.D. (N = 3).

Figure 3

DNA ends and homologous recombination: one-ended versus two-ended DSBs. (A) Sister chromatid exchange. Replication fork collapse leads to a partially replicated chromosome with a one-ended DSB, which can invade the intact sister to restart the fork. As there is only one double-stranded end per fork collapse, ligation mediated by NHEJ proteins would not be predicted to be involved in its repair, and Ku would not block entry of the HDR machinery (blue circle). (B) Gene targeting. A linearized gene-targeting substrate involves the homologous invasion of a one-ended DSB with an intact chromosome, similar to SCE formation in A, but in gene targeting there is a one-ended DSB on each side of the targeting fragment. NHEJ-mediated ligation does not appear to be competitive with this reaction in mammalian cells, possibly because Ku is nonblocking for HDR proteins at these one-ended DSBs. (C) Classical DSB with two ends. I-SceI endonuclease generates a two-ended DSB, with the ends in close proximity. The NHEJ proteins (red ovals) mediate a ligation reaction that can compete with the HDR machinery. Even in the absence of an ability to ligate the ends (e.g., by loss of XRCC4 or DNA-PKcs), Ku can interact with the two-ended DSB and block entry by HDR proteins.