Analysis of the Role of Homology Arms in Gene-Targeting Vectors in Human Cells

<div><p>Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4), the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.</p></div

NHEJ-independent random integration is significantly decreased when a homology arm is deleted from the <i>HPRT</i> targeting vector.

<p>(A) Integration frequency of pHPRT8.9-Puro vectors and their derivatives in Nalm-6 wild-type and <i>LIG4</i>-null cells. The ratio of integration frequency in <i>LIG4</i>-null to wild-type cells is indicated in the right column. At least five independent experiments were performed for each vector. The data for the arm-proficient vectors are the same as that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108236#pone-0108236-g002" target="_blank">Figure 2D</a>. (B) Integration frequency of pHPRT2.2-Puro vectors and their derivatives in Nalm-6 wild-type and <i>LIG4</i>-null cells. The ratio of integration frequency in <i>LIG4</i>-null to wild-type cells is indicated in the right column. At least three independent experiments were performed for each vector. Symbols are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108236#pone-0108236-g002" target="_blank">Figure 2D</a>, and the data for the arm-proficient vectors are the same as that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108236#pone-0108236-g002" target="_blank">Figure 2D</a>.</p

The short-arm vector pHPRT2.2-Puro(−) functions as a genuine targeting vector.

<p>(A) Gene-targeting efficiency of pHPRT8.9-Puro(−) and pHPRT2.2-Puro(−) in wild-type and <i>LIG4</i>-null cells. (B) Random and targeted integration frequencies of pHPRT8.9-Puro(−) and pHPRT2.2-Puro(−) in wild-type and <i>LIG4</i>-null cells. At least three independent experiments were performed for each vector.</p

Gene targeting is inefficient in human somatic cells.

<p>When targeting vector is transfected into human cells, random integration by non-homologous recombination occurs at least 2 to 3 orders of magnitude more frequently than homologous recombination-mediated targeted integration. The LIG4-dependent NHEJ pathway has been thought to be responsible for random integration, but recent evidence indicates a contribution from LIG4-independent mechanisms that rely on LIG1/3 (DNA ligase I or IIIα). The gene-targeting efficiency is calculated by dividing the number of targeted clones with that of drug-resistant clones analyzed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108236#s3" target="_blank">Materials and Methods</a> for details).</p

<i>HPRT</i> targeting vectors with long, but not short, homology arms stimulate NHEJ-independent random integration.

<p>(A) Schematic representation of <i>HPRT</i> targeting vectors pHPRT8.9-Puro(+) and pHPRT8.9-Puro(−). (B) Schematic representation of <i>HPRT</i> targeting vectors pHPRT2.2-Puro(+) and pHPRT2.2-Puro(−). (C) Structural features of the <i>HPRT</i> targeting vectors. (D) Integration frequency of <i>HPRT</i> targeting vectors and pPGK-Puro (a non-targeting vector) in human Nalm-6 wild-type and <i>LIG4</i>-null cells. The ratio of integration frequency in <i>LIG4</i>-null to wild-type cells is indicated in the right column. At least six independent experiments were performed for each vector. Note that pPGK-Puro harbors little or no homology to the human genome. Grey lines indicate the lengths of plasmid backbones, and § denotes a 14-bp sequence.</p

Additional file 1 of Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection

Table S1. Oligonucleotides used in this study. Figure S1. Construction of pENTR lox71-P. Figure S2. Construction of pDEST SA-IRES-DTA-pA. Figure S3. Schematic representation of exon-trapping gene targeting at the mouse Rosa26 and human HPRT loci

DNA Ligase IV and Artemis Act Cooperatively to Suppress Homologous Recombination in Human Cells: Implications for DNA Double-Strand Break Repair

<div><p>Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.</p></div

Loss of Artemis alleviates hypersensitivity of <i>LIG4</i>^−/− cells to low-dose irradiation- and Top2-induced DSBs.

<p>(A–D) Sensitivity of <i>wild-type</i>, <i>ARTEMIS</i>^−/−, <i>LIG4</i>^−/−, and <i>LIG4</i>^−/−<i>ARTEMIS</i>^−/− cells to etoposide (A), X-rays (B and, for low-dose range, C), and neocarzinostatin (D), as determined by clonogenic assays. Shown are the mean ± SD of at least three independent experiments. Where absent, error bars fall within symbols. (E) Average number of γ-H2AX foci per cell. γ-H2AX focus-formation assay was performed using 1 Gy-irradiated cells.</p

Genetic deletion of human DNA ligase IV confers resistance to killing by replication-associated DSBs.

<p>(A) Schematic representation of replication-associated DSBs. SSBs accumulate in the genome when cells are treated with CPT or NU1025 (a). In mammalian cells, SSBs are primarily repaired by the SSB repair pathway (b). If left unrepaired, however, SSBs are converted into DSBs, accompanied by fork collapse, upon collision with replication forks (c,d), and repaired by the DSB repair mechanism (e). (B) Detection of γ -H2AX using cells treated with MMS, neocarzinostatin, etoposide, or camptothecin as described in Materials and Methods. NT, untreated cells. (C, D) Sensitivity of <i>wild-type</i>, <i>RAD54</i>^−/−, <i>LIG4</i>^−/−, and <i>LIG4</i>^−/−<i>RAD54</i>^−/− cells to CPT (C) and NU1025 (D), as determined by growth inhibition assays. Data are the mean ± SD of at least three independent experiments. Where absent, error bars fall within symbols. (E) Relative caspase-3/7 activity after CPT treatment. (F) Western blot analysis for p53. Twenty micrograms of whole cell extract from wild-type (WT) and mutant cell lines were loaded on a 7.5% SDS-polyacrylamide gel.</p

Model for DSB repair control in human somatic cells.

<p>Cells can suffer various types of DSBs, involving those induced by irradiation, Top2 (“direct DSBs”) and, in S phase, replication fork collapse (“indirect” DSBs). The Ku70/Ku80 complex, Ku, can rapidly bind to most, if not all, DSBs to initiate an NHEJ reaction. Ku-unbound DSBs, if any, are not subjected to NHEJ, but can be repaired by HR or alternative end-joining (AltEJ) pathways <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072253#pone.0072253-Corneo1" target="_blank">[58]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072253#pone.0072253-Yan1" target="_blank">[60]</a>. Irrespective of accuracy, those repair events lead to cellular survival, with the exception that NHEJ after indirect DSBs is toxic and leads to cell death. Loss of DNA ligase IV completely abolishes NHEJ and shunts the DSB toward HR or AltEJ, or may result in cell death <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072253#pone.0072253-Adachi2" target="_blank">[18]</a>. Loss of Artemis, on the other hand, does not completely block the NHEJ reaction, but would more efficiently shunt the DSB to HR. Similar situations (i.e., some Ku-bound DSBs are not mended by NHEJ) can be caused by incomplete end-trimming reactions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072253#pone.0072253-Ma2" target="_blank">[61]</a> and/or when the cell has a huge number of DSBs. When NHEJ is unsuccessful at rejoining the DSB, the cell would give up the abortive NHEJ reaction by somehow relieving the Artemis-mediated HR suppression. See text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072253#pone.0072253.s007" target="_blank">Figure S7</a> for further details.</p