XRCC3 Controls the Fidelity of Homologous Recombination Mark A Brenneman, Brant M Wagener, Cheryl A Miller, Chris Allen, Jac A Nickoloff  Molecular Cell  Volume 10, Issue 2, Pages 387-395 (August 2002) DOI: 10.1016/S1097-2765(02)00595-6

Figure 1 HR Substrate and Products A neo direct repeat HR substrate is present as a single integrated copy in irs-SF 1822/1823 cells. The recipient neo allele (MMTVneo) is driven by the mouse mammary tumor virus LTR promoter/enhancer, but inactivated by an I-SceI recognition sequence inserted into the natural BanII site. The donor neo allele (neo12) has no promoter and contains 12 silent RFLP markers (shading). The neo genes flank an SV40 promoter-driven E. coli guanosine phosphotransferase gene (SVgpt). EcoRI sites (RI) flank the neo repeats; EcoRV sites (RV) occur once in SVgpt and in genomic DNA (thick lines) outside the HR substrate. Gene conversion without crossover transfers neo12 markers to MMTVneo but preserves the gross substrate structure. Intrachromosomal conversion with crossover, unequal sister chromatid exchange, single-strand annealing, or break-induced replication deletes DNA between the neo genes. In all cases, the I-SceI site is replaced by BanII. Events other than HR (i.e., NHEJ, chromosome loss) are not detected. PCR primers A, B, and C were used to amplify MMTVneo (see Experimental Procedures). Molecular Cell 2002 10, 387-395DOI: (10.1016/S1097-2765(02)00595-6)

Figure 2 Chromosome Rearrangements Associated with HR Southern analysis of representative G418r products from XRCC3-complemented (A) and noncomplemented (B) irs1-SF strain 1822 cells. The position of the 10.5 kbp EcoRI fragment band was determined from a control lane with EcoRI-digested pMSGneo2S12HIS (data not shown). (C) EcoRV patterns from representative rearranged products of strain 1822. Simple gene conversion products (data not shown) give EcoRV fragments GC #1 and #2. A1–A4 indicate additional fragments seen in rearranged products. Molecular Cell 2002 10, 387-395DOI: (10.1016/S1097-2765(02)00595-6)

Figure 3 Gene Conversion Tract Spectra Individual HR products were classified according to the RFLP markers converted. Black bars represent converted markers. The minimum hDNA region is defined by the most distal converted markers. Data are shown for complemented (+) and noncomplemented (−) strain 1822. Values in parentheses indicate the number of XRCC3− products with rearrangements/total number of that tract type. Data for wild-type CHO cells (WT) are from Taghian and Nickoloff (1997). Product types 1–25 are continuous, and D1–D6 are discontinuous. The total number of mapped products from each strain is given below. Molecular Cell 2002 10, 387-395DOI: (10.1016/S1097-2765(02)00595-6)

Figure 4 Average Tract Lengths and Frequencies of Discontinuous Tracts (A) Average tract lengths for wild-type CHO and 1822 cells were calculated from data in Figure 3, and for 1823 cells from data not shown. Average tract length in XRCC3− 1822 cells was significantly longer than both wild-type and complemented 1822 cells (indicated by **). The increased tract length was apparent in both the full 1822 product set (black bar) and the subset that had undergone simple gene conversion (hatched bar). The average tract length in XRCC3− 1823 cells was significantly longer than wild-type cells, but not complemented 1823 cells (indicated by *). (B) Percent discontinuous tracts for wild-type CHO, 1822, and 1823 cells with or without XRCC3 complementation. Discontinuous tracts were significantly more frequent in XRCC3− 1822 and 1823 cells than both complemented and wild-type cells (indicated by **). Among the subset of simple conversion products of strain 1822 products, discontinuous tracts were significantly more frequent than in wild-type (p = 0.05) but not complemented 1822 cells (p = 0.07) owing to small sample size (indicated by *). Molecular Cell 2002 10, 387-395DOI: (10.1016/S1097-2765(02)00595-6)

Figure 5 Frequencies of Marker Inclusion in hDNA Percent conversion of each marker as a function of distance from the DSB was calculated from data in Figure 3. Statistical analysis was performed for each marker between the full XRCC3− data set and complemented 1822 cells: **p ≤ 0.0002; *p < 0.01, Fisher exact tests. Similar results were obtained with the subset of XRCC3− products that arose by simple conversion, with significant differences (p ≤ 0.04) at all markers that showed differences with the full data set except for B733 (located at −100 bp from I-SceI). Molecular Cell 2002 10, 387-395DOI: (10.1016/S1097-2765(02)00595-6)

Figure 6 Potential Roles for XRCC3 Early and Late in HR DSBs are processed by NHEJ or HR. Nuclease-induced DSBs can be rejoined precisely, or imprecisely, leading to deletions, insertions, and chromosome translocations. XRCC3 deficiency might compromise HR efficiency and fidelity at any of three steps. XRCC3 might promote formation, or stabilize RAD51 nucleoprotein filaments (step 1) or strand invasion (step 2). Failure at these steps shunts DSBs from HR to NHEJ. XRCC3 might promote second strand invasion (step 3), and failure at this point could result in mixed HR/NHEJ products that appear as local rearrangements associated with HR. Finally, XRCC3 may stabilize the hDNA intermediate. The second end may invade even in the absence of XRCC3, and in this case, if long hDNA regions are formed, the intermediates may be stable enough to be resolved as HR products, albeit with long and/or discontinuous tracts. This model is not meant to be restrictive. For example, HR may not require two-ended invasions, and XRCC3 might be required for the formation or proper resolution of single-end invasion intermediates. Molecular Cell 2002 10, 387-395DOI: (10.1016/S1097-2765(02)00595-6)