Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks.

Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal

Cell Cycle-Dependent Induction of Homologous Recombination by a Tightly Regulated I-SceI Fusion Protein

Double-strand break repair is executed by two major repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). Whereas NHEJ contributes to the repair of ionizing radiation (IR)-induced double strand breaks (DSBs) throughout the cell cycle, HR acts predominantly during the S and G2 phases of the cell cycle. The rare-cutting restriction endonuclease, I-SceI, is in common use to study the repair of site-specific chromosomal DSBs in vertebrate cells. To facilitate analysis of I-SceI-induced DSB repair, we have developed a stably expressed I-SceI fusion protein that enables precise temporal control of I-SceI activation, and correspondingly tight control of the timing of onset of site-specific chromosome breakage. I-SceI-induced HR showed a strong, positive linear correlation with the percentage of cells in S phase, and was negatively correlated with the G1 fraction. Acute depletion of BRCA1, a key regulator of HR, disrupted the relationship between S phase fraction and I-SceI-induced HR, consistent with the hypothesis that BRCA1 regulates HR during S phase

XRCC2 and XRCC3 Regulate the Balance between Short- and Long-Tract Gene Conversions between Sister Chromatids▿ §

Sister chromatid recombination (SCR) is a potentially error-free pathway for the repair of DNA lesions associated with replication and is thought to be important for suppressing genomic instability. The mechanisms regulating the initiation and termination of SCR in mammalian cells are poorly understood. Previous work has implicated all the Rad51 paralogs in the initiation of gene conversion and the Rad51C/XRCC3 complex in its termination. Here, we show that hamster cells deficient in the Rad51 paralog XRCC2, a component of the Rad51B/Rad51C/Rad51D/XRCC2 complex, reveal a bias in favor of long-tract gene conversion (LTGC) during SCR. This defect is corrected by expression of wild-type XRCC2 and also by XRCC2 mutants defective in ATP binding and hydrolysis. In contrast, XRCC3-mediated homologous recombination and suppression of LTGC are dependent on ATP binding and hydrolysis. These results reveal an unexpectedly general role for Rad51 paralogs in the control of the termination of gene conversion between sister chromatids

Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells

A proportion of homologous recombination (HR) events in mammalian cells resolve by “long tract” gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer

Complex Breakpoints and Template Switching Associated with Non-canonical Termination of Homologous Recombination in Mammalian Cells.

A proportion of homologous recombination (HR) events in mammalian cells resolve by "long tract" gene conversion, reflecting copying of several kilobases from the donor sister chromatid prior to termination. Cells lacking the major hereditary breast/ovarian cancer predisposition genes, BRCA1 or BRCA2, or certain other HR-defective cells, reveal a bias in favor of long tract gene conversion, suggesting that this aberrant HR outcome might be connected with genomic instability. If termination of gene conversion occurs in regions lacking homology with the second end of the break, the normal mechanism of HR termination by annealing (i.e., homologous pairing) is not available and termination must occur by as yet poorly defined non-canonical mechanisms. Here we use a previously described HR reporter to analyze mechanisms of non-canonical termination of long tract gene conversion in mammalian cells. We find that non-canonical HR termination can occur in the absence of the classical non-homologous end joining gene XRCC4. We observe obligatory use of microhomology (MH)-mediated end joining and/or nucleotide addition during rejoining with the second end of the break. Notably, non-canonical HR termination is associated with complex breakpoints. We identify roles for homology-mediated template switching and, potentially, MH-mediated template switching/microhomology-mediated break-induced replication, in the formation of complex breakpoints at sites of non-canonical HR termination. This work identifies non-canonical HR termination as a potential contributor to genomic instability and to the formation of complex breakpoints in cancer

Breakpoints of non-canonical LTGC termination in five <i>XRCC4</i>^fl/fl and two <i>XRCC4</i>^Δ/Δ clones.

<p>Cartoon shows approximate positions of breakpoints. Black numbers mark site of LTGC termination; paired blue numbers mark extent of second end resection for the same clone (not to scale). Numbers correlate with the numbered clones in lower panel, showing length of gene conversion tract (black) and extent of second end resection (blue) in each clone, with genotype as indicated. Red nucleotides: N-insertions at the breakpoint. Dual black/blue nucleotide sequences at the breakpoint represent microhomology.</p

Restriction mapping of products of non-canonical LTGC termination.

<p>Genomic DNA from two clones in which LTGC was terminated by non-canonical mechanisms was digested with the restriction enzymes shown and analyzed by Southern blotting (<i>GFP</i> probe). Restriction enzymes used were SacI (Sa), HindIII (H), BamHI (B), EcoRI (E) and SpeI (Sp). Cartoons on right show restriction fragment sizes observed for HR reporter at the <i>ROSA26</i> locus. The presence or absence of the 3.2kb amplification product in each restriction digest helps to localize the site of LTGC termination within the reporter. (A) <i>XRCC4</i>^fl/fl clone in which termination of LTGC occurred between HindIII and EcoRI sites within the HR reporter. EcoRI and SpeI digests lack the 3.2kb amplification product. (B) <i>XRCC4</i>^Δ/Δ clone in which termination of LTGC occurred between SacI and HindIII sites within the HR reporter. HindIII, EcoRI and SpeI digests lack the 3.2kb amplification product. In this clone, the right hand arms of the SpeI and HindIII digests are much smaller (SpeI) or larger (HindIII) than would be predicted. This is explained by the deletion of ~3.5kb from the second end of the DSB, as revealed by sequencing (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006410#pgen.1006410.g006" target="_blank">Fig 6B</a>).</p

MMBIR model of complex breakpoint shown in Fig 5B.

<p>Strand separation occurs within the DNA of the second end of the break ~3.5 kb from the I-SceI site. One possible source depicted here is a stalled replication fork. The pale orange and blue arrows flanking the stalled fork represent the exposed ssDNA sequences that template the inversion (orange) and inversion-duplication (blue) sequences identified within the LTGC breakpoint (A) The displaced nascent strand product of LTGC (black) acquires a ≥21bp insertion (red; whether templated or untemplated is unknown). (B) Microhomology-mediated base-pairing between the 3’ end of the displaced nascent strand and ssDNA of the stalled replication fork. (C) The lagging strand template enables retrograde nascent strand extension (“MMBIR”), generating the inversion sequences as shown. (D) Displacement of the nascent strand. (E) Four base pair MH-mediated (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006410#pgen.1006410.g006" target="_blank">Fig 6B</a>) annealing of the 3’ end of the displaced nascent strand with the 5’ end of the duplicated region on the leading strand. Black arrowheads: sites of endonucleolytic cleavage that would enable completion of rearrangement by MMEJ-mediated rejoining. Alternatively, more extensive MMBIR copying could complete the rearrangement.</p

I-SceI-induced LTGC products in <i>XRCC4</i>^fl/fl and <i>XRCC4</i>^Δ/Δ cells.

<p>I-SceI-induced LTGC products in <i>XRCC4</i>^fl/fl and <i>XRCC4</i>^Δ/Δ cells.</p