DNA Ligase 1 is an essential mediator of sister chromatid telomere fusions in G2 cell cycle phase

Fusion of critically short or damaged telomeres is associated with the genomic rearrangements that support malignant transformation. We have demonstrated the fundamental contribution of DNA ligase 4-dependent classical non-homologous end-joining to long-range inter-chromosomal telomere fusions. In contrast, localized genomic recombinations initiated by sister chromatid fusion are predominantly mediated by alternative non-homologous end-joining activity that may employ either DNA ligase 3 or DNA ligase 1. In this study, we sought to discriminate the relative involvement of these ligases in sister chromatid telomere fusion through a precise genetic dissociation of functional activity. We have resolved an essential and non-redundant role for DNA ligase 1 in the fusion of sister chromatids bearing targeted double strand DNA breaks that is entirely uncoupled from its requisite engagement in DNA replication. Importantly, this fusogenic repair occurs in cells fully proficient for non-homologous end-joining and is not compensated by DNA ligases 3 or 4. The dual functions of DNA ligase 1 in replication and non-homologous end-joining uniquely position and capacitate this ligase for DNA repair at stalled replication forks, facilitating mitotic progression

FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response

Fanconi anemia (FA) is an inherited cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA proteins act in a linear hierarchy: following ICL detection on chromatin, the FA core complex monoubiquitinates and recruits the central FANCI and FANCD2 proteins that subsequently coordinate ICL removal and repair of the ensuing DNA double-stranded break by homology-dependent repair (HDR). FANCD2 also functions during the replication stress response by mediating the restart of temporarily stalled replication forks thereby suppressing the firing of new replication origins. To address if FANCI is also involved in these FANCD2-dependent mechanisms, we generated isogenic FANCI-, FANCD2- and FANCI:FANCD2 double-null cells. We show that FANCI and FANCD2 are partially independent regarding their protein stability, nuclear localization and chromatin recruitment and contribute independently to cellular proliferation. Simultaneously, FANCD2-but not FANCI-plays a major role in HDR-mediated replication restart and in suppressing new origin firing. Consistent with this observation, deficiencies in HDR-mediated DNA DSB repair can be overcome by stabilizing RAD51 filament formation in cells lacking functional FANCD2. We propose that FANCI and FANCD2 have partially non-overlapping and possibly even opposing roles during the replication stress response

The Mechanism of Gene Targeting in Human Somatic Cells

<div><p>Gene targeting in human somatic cells is of importance because it can be used to either delineate the loss-of-function phenotype of a gene or correct a mutated gene back to wild-type. Both of these outcomes require a form of DNA double-strand break (DSB) repair known as homologous recombination (HR). The mechanism of HR leading to gene targeting, however, is not well understood in human cells. Here, we demonstrate that a two-end, ends-out HR intermediate is valid for human gene targeting. Furthermore, the resolution step of this intermediate occurs via the classic DSB repair model of HR while synthesis-dependent strand annealing and Holliday Junction dissolution are, at best, minor pathways. Moreover, and in contrast to other systems, the positions of Holliday Junction resolution are evenly distributed along the homology arms of the targeting vector. Most unexpectedly, we demonstrate that when a meganuclease is used to introduce a chromosomal DSB to augment gene targeting, the mechanism of gene targeting is inverted to an ends-in process. Finally, we demonstrate that the anti-recombination activity of mismatch repair is a significant impediment to gene targeting. These observations significantly advance our understanding of HR and gene targeting in human cells.</p></div

Gene targeting is marked by a characteristic SNP retention signature.

<p>(A) The rAAV targeting vector. The NEO cassette (white rectangle) is flanked by the homology armss (green and blue rectangles). NdeI, EcoRI, NcoI, AseI, SspI, SacI, XbaI and SbfI represent vector-specific restriction sites created by SNPs. LHP/RHP represent vector-specific palindromes (lollipops) created by introducing SNPs. The flanking hairpins represent inverted terminal repeats. (B and C) The HPRT locus before and after gene targeting. The NEO cassette replaces exon 3 (grey) of HPRT upon gene targeting. The theoretical positions of the viral markers are indicated in bold vertical lines and (?) symbols. The arrows represent PCR primers. P1xP3 and P4xP6 amplify the left and right homology arms of the gene targeted clones, respectively, and P2xP3 and P4xP5 amplify the homology arms of the randomly integrated clones, respectively. The LHP destroys a chromosomal BbvCI restriction site upon integration. (D) The dsDNA targeting vector. All symbols are defined above. (E, F, G and H) SNP retention signatures of rAAV gene targeting for HCT116 and DLD-1 cell lines, plasmid dsDNA gene targeting and random integration, respectively. The distance to the central heterology (cartooned as a vertical black line) is calculated from the inner ends of the homology arms. Markers on the left homology arms are indicated with negative distances. Green and purple lines represent the linear regression between the retention frequency and the distance of the viral markers for the left and right homology arms, respectively.</p

Models for rAAV gene targeting.

<p>(A) The HR model. Black, red and blue lines correspond to genomic DNA, viral and genomic homology arms, respectively; the bold green line corresponds to the selection cassette. The vertical arrows imply that the viral DNA becomes double-stranded and the inverted terminal repeats are processed before integrating into the genome. Open arrows represent the sites of HJ cleavage and ligation. A sectored colony is formed during mitosis. (B) The single strand assimilation model. All symbols are as in (A). The virus that anneals to the genomic DNA is single-stranded. In the ensuing mitosis, an unsectored colony is formed under drug selection.</p

Models for rAAV gene targeting in the presence of DSBs.

<p>(A) rAAV gene targeting in the presence of DSBs. Dotted lines and arrowheads correspond to de novo DNA synthesis, which is color-coded to match the templates. Orange slashes represent half I-SceI sites and the lightening bolt represents I-SceI-induced cleavage. All other symbols are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004251#pgen-1004251-g002" target="_blank">Figure 2</a>. The chromosomal ends are processed and invade the viral DNA in an ends-in configuration. Two predicted SNP retention patterns (minus and plus drug selection, respectively) are cartooned as (1) and (2), respectively. (B) Holliday Junction dissolution. Branch migration forces the HJs towards the drug selection cassette and the HJ is cancelled. The predicted SNP retention pattern is cartooned in (3). (C) Synthesis dependent strand annealing. If the synapsed structure shown in (A) collapses, recombination can still occur by SDSA. This mechanism, like HJ dissolution (B), predicts the SNP retention pattern shown in (3).</p

rAAV gene targeting is suppressed in a mismatch repair-proficient background.

<p>(A) The rAAV targeting vectors. All symbols as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004251#pgen-1004251-g001" target="_blank">Figure 1A</a>. 2SNPs and 15SNPs indicate the total number of mismatches in the vectors. (B) Effects of mismatches and the host mismatch repair status on rAAV gene targeting. The rAAV gene targeting efficiency is expressed as the ratio of correctly targeted clones divided by the sum of the correctly targeted plus the randomly integrated clones. All results are normalized to the parental (MLH1⁻) cell line. The mean ± SEM of three independent experiments is shown. The MLH1 expression in the parental and MLH1⁺ cell lines is shown in the inserted Western blot panel. b-actin was used as a loading control. (C and D) The SNP retention signature of rAAV gene targeting and random integration respectively in the mismatch repair (MLH1⁺) -proficient background. All symbols are as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004251#pgen-1004251-g001" target="_blank">Figure 1E</a>.</p

rAAV gene targeting is associated with the formation of sectored colonies.

<p>Solid boxes on the top (not to scale) represents diagnostic markers on the virus (blue) and genomic DNA (yellow). The numbers indicate the actual positions of the markers. The NEO cassette and exon 3 of HPRT are indicated in white and grey, respectively. Each line on the bottom corresponds to an independent gene targetin event. The blue, yellow and green segments are color-coded to represent viral, genomic and sectored tracts, respectively. The top and bottom panels show results obtained from HCT116 and DLD-1 cells, respectively.</p

The HSV-1 exonuclease, UL12, stimulates recombination by a single strand annealing mechanism.

Production of concatemeric DNA is an essential step during HSV infection, as the packaging machinery must recognize longer-than-unit-length concatemers; however, the mechanism by which they are formed is poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication involves recombination-dependent replication reminiscent of bacteriophages λ and T4. Similar to λ, HSV-1 encodes a 5'-to-3' exonuclease (UL12) and a single strand annealing protein [SSAP (ICP8)] that interact with each other and can perform strand exchange in vitro. By analogy with λ phage, HSV may utilize viral and/or cellular recombination proteins during DNA replication. At least four double strand break repair pathways are present in eukaryotic cells, and HSV-1 is known to manipulate several components of these pathways. Chromosomally integrated reporter assays were used to measure the repair of double strand breaks in HSV-infected cells. Single strand annealing (SSA) was increased in HSV-infected cells, while homologous recombination (HR), non-homologous end joining (NHEJ) and alternative non-homologous end joining (A-NHEJ) were decreased. The increase in SSA was abolished when cells were infected with a viral mutant lacking UL12. Moreover, expression of UL12 alone caused an increase in SSA, which was completely eliminated when a UL12 mutant lacking exonuclease activity was expressed. UL12-mediated stimulation of SSA was decreased in cells lacking the cellular SSAP, Rad52, and could be restored by coexpressing the viral SSAP, ICP8, indicating that an SSAP is also required. These results demonstrate that UL12 can specifically stimulate SSA and that either ICP8 or Rad52 can function as an SSAP. We suggest that SSA is the homology-mediated repair pathway utilized during HSV infection