Dataset Information

Depletion of Werner helicase results in mitotic hyperrecombination and pleiotropic homologous and nonhomologous recombination phenotypes.

ABSTRACT: Werner syndrome (WS) is a rare, segmental progeroid syndrome caused by defects in the WRN gene, which encodes a RecQ helicase. WRN has roles in many aspects of DNA metabolism including DNA repair and recombination. In this study, we exploited two different recombination assays previously used to describe a role for the structure-specific endonuclease ERCC1-XPF in mitotic and targeted homologous recombination. We constructed Chinese hamster ovary (CHO) cell lines isogenic with the cell lines used in these previous studies by depleting WRN using shRNA vectors. When intrachromosomal, mitotic recombination was assayed in WRN-depleted CHO cells, a hyperrecombination phenotype was observed, and a small number of aberrant recombinants were generated. Targeted homologous recombination was also examined in WRN-depleted CHO cells using a plasmid-chromosome targeting assay. In these experiments, loss of WRN resulted in a significant decrease in nonhomologous integration events and ablation of recombinants that required random integration of the corrected targeting vector. Aberrant recombinants were also recovered, but only from WRN-depleted cells. The pleiotropic recombination phenotypes conferred by WRN depletion, reflected in distinct homologous and nonhomologous recombination pathways, suggest a role for WRN in processing specific types of homologous recombination intermediates as well as an important function in nonhomologous recombination.

SUBMITTER: Rahn JJ

PROVIDER: S-EPMC2949496 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Depletion of Werner helicase results in mitotic hyperrecombination and pleiotropic homologous and nonhomologous recombination phenotypes.

Rahn Jennifer J JJ Lowery Megan P MP Della-Coletta Luis L Adair Gerald M GM Nairn Rodney S RS

Mechanisms of ageing and development 20100812 9

Werner syndrome (WS) is a rare, segmental progeroid syndrome caused by defects in the WRN gene, which encodes a RecQ helicase. WRN has roles in many aspects of DNA metabolism including DNA repair and recombination. In this study, we exploited two different recombination assays previously used to describe a role for the structure-specific endonuclease ERCC1-XPF in mitotic and targeted homologous recombination. We constructed Chinese hamster ovary (CHO) cell lines isogenic with the cell lines used ...[more]

PMID: 20708636

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Project description:Four unrelated families with the same unbalanced translocation der(4)t(4;11)(p16.2;p15.4) were identified. Both of the breakpoint regions in 4p16.2 and 11p15.4 were narrowed to large ~359-kb and ~215-kb low-copy repeat (LCR) clusters, respectively, by aCGH and SNP array analyses. DNA sequencing enabled mapping the breakpoints of one translocation to 24-bp within interchromosomal paralogous LCRs of ~130-kb in length and 94.7% DNA sequence identity located in olfactory receptor gene clusters, indicating nonallelic homologous recombination (NAHR) as the mechanism for translocation. To investigate the potential involvement of interchromosomal LCRs in recurrent chromosomal translocation formation, we performed computational genome-wide analyses and identified 5292 interchromosomal LCR substrate pairs, > 5-kb in size and sharing > 94% sequence identity that can potentially mediate chromosomal translocations. Additional proof for interchromosomal NAHR mediated translocation formation was provided by sequencing the breakpoints of another recurrent translocation, der(8)t(8;12)(p23.1;p13.31). The NAHR sites were mapped within 55-bp in ~7.8-kb paralogous subunits of 95.3% sequence identity located in the ~579-kb (chr8) and ~287-kb (chr12) LCR clusters. We demonstrate that NAHR mediates recurrent constitutional translocations throughout the human genome and provide a computationally determined genome-wide “recurrent translocation map”. Affymetrix Genome-Wide SNP Array 6.0 arrays (Affymetrix, Santa Clara, California) were employed to define the breakpoints on chromosomes 4, 8, 11, and 12. Analysis was performed according to the Genome-Wide Human SNP Nsp/Sty Assay Kit 5.0/6.0 protocol provided by the supplier. The arrays were scanned using a GeneChip® Scanner 3000 7G (Affymetrix, Inc.) and results were analyzed using Genotyping Console version 2.1 software. patient 1: Version 5 BAC array contained 853 BAC/PAC clones designed to cover genomic regions of 75 known genomic disorders, all 41 subtelomeric regions, and 43 pericentromeric regions. For each patient sample, two experiments were performed with reversal of the dye labels for the control and test samples. patient 2: Version 5.1 BAC array contained 853 BAC/PAC clones designed to cover genomic regions of 75 known genomic disorders, all 41 subtelomeric regions, and 43 pericentromeric regions. For each patient sample, two experiments were performed with reversal of the dye labels for the control and test samples. patient 3: The BAC emulated Version 6.1 OLIGO array was comprised of approximately 42,460 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). The 42.46K oligonucleotides (oligos) were selected from initial testing of 105,000 oligos derived from the Agilent eArray library with strict selection criteria and removal of repetitive sequences to ensure optimal performance with greater dynamic range. This targeted 42.46 K OLIGO array (V6 OLIGO) corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. patient 5: The BAC emulated Version 6.3 OLIGO array was comprised of approximately 43,580 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). This targeted OLIGO array corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. patient 6: The BAC emulated Version 6.4 OLIGO array was comprised of approximately 43,700 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). This targeted OLIGO array corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. Patient 4 was a known t(4;11) translocation patient form previous report and only ran on Affymetrix array.

Dataset Information

Depletion of Werner helicase results in mitotic hyperrecombination and pleiotropic homologous and nonhomologous recombination phenotypes.

Publications

Depletion of Werner helicase results in mitotic hyperrecombination and pleiotropic homologous and nonhomologous recombination phenotypes.

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