Pervasive migration of organellar DNA to the nucleus in plants

A surprisingly large number of plant nuclear DNA sequences inferred to be remnants of chloroplast and mitochondrial DNA migration events were detected through computer-assisted database searches. Nineteen independent organellar DNA insertions, with a median size of 117 by (range of 38 to >785 bp), occur in the proximity of 15 nuclear genes. One fragment appears to have been passed through a RNA intermediate, based on the presence of an edited version of the mitochondrial gene in the nucleus. Tandemly arranged fragments from disparate regions of organellar genomes and from different organellar genomes indicate that the fragments joined together from an intracellular pool of RNA and/or DNA before they integrated into the nuclear genome. Comparisons of integrated sequences to genes lacking the insertions, as well as the occurrence of coligated fragments, support a model of random integration by end joining. All transferred sequences were found in noncoding regions, but the positioning of organellar-derived DNA in introns, as well as regions 5 and 3 to nuclear genes, suggests that the random integration of organellar DNA has the potential to influence gene expression patterns. A semiquantitative estimate was performed on the amount of organellar DNA being transferred and assimilated into the nucleus. Based on this database survey, we estimate that 3–7% of the plant nuclear genomic sequence files contain organellar-derived DNA. The timing and the magnitude of genetic flux to the nuclear genome suggest that random integration is a substantial and ongoing process for creating sequence variation.Correspondence to: J.L. Blanchard     

A role for protein phosphatase 4 in regulating non-homologous end-joining

Comment on: Liu J, et al. Cell Cycle 2012; 11:2643-9.     

Remodeling and spacing factor 1 (RSF1) deposits centromere proteins at DNA double-strand breaks to promote non-homologous end-joining

The cellular response to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in native chromatin requires a tight coordination between the activities of DNA repair machineries and factors that modulate chromatin structure. SMARCA5 is an ATPase of the SNF2 family of chromatin remodeling factors that has recently been implicated in the DSB response. It forms distinct chromatin remodeling complexes with several non-canonical subunits, including the remodeling and spacing factor 1 (RSF1) protein. Despite the fact that RSF1 is often overexpressed in tumors and linked to tumorigenesis and genome instability, its role in the DSB response remains largely unclear. Here we show that RSF1 accumulates at DSB sites and protects human cells against IR-induced DSBs by promoting repair of these lesions through homologous recombination (HR) and non-homologous end-joining (NHEJ). Although SMARCA5 regulates the RNF168-dependent ubiquitin response that targets BRCA1 to DSBs, we found RSF1 to be dispensable for this process. Conversely, we found that RSF1 facilitates the assembly of centromere proteins CENP-S and CENP-X at sites of DNA damage, while SMARCA5 was not required for these events. Mechanistically, we uncovered that CENP-S and CENP-X, upon their incorporation by RSF1, promote assembly of the NHEJ factor XRCC4 at damaged chromatin. In contrast, CENP-S and CENP-X were dispensable for HR, suggesting that RSF1 regulates HR independently of these centromere proteins. Our findings reveal distinct functions of RSF1 in the 2 major pathways of DSB repair and explain how RSF1, through the loading of centromere proteins and XRCC4 at DSBs, promotes repair by non-homologous end-joining.     

Characteristics of the end-joining of DNA double-strand breaks by the ataxia-telangiectasia nuclear extract

A double-strand break was introduced in plasmid pZErO-2 at a specific site within the ccdB gene that is lethal to Escherichia coli cells and treated with nuclear extracts from human cells. The efficiency of rejoining was monitored by Southern blot analysis and the fidelity of rejoining was measured by expressing the ccdB gene after bacterial transformation. The efficiency of rejoining in the nuclear extract from an ataxia-telangiectasia (A-T) cell line was comparable to that from a control cell line. However, the accuracy of rejoining was much lower for the A-T cell extract than for the control cell extract. All mutations were deletions, most of which contained short direct repeats at the breakpoint junctions. The deletion spectrum caused by the A-T nuclear extract was distinct from that of the control extract. These results indicate that the ccdB gene is useful for analysis of mis-rejoining and that A-T cells have certain deficiencies in end-joining of double-strand breaks in DNA.     

Involvement of the insulin-like growth factor binding proteins in the cancer cell response to DNA damage

The complex mechanisms that cells have evolved to meet the challenge of constant exposure to DNA-damaging stimuli, also serve to protect cancer cells from the cytotoxic effects of chemo- and radiotherapy. IGFBPs appear to be involved, directly or indirectly, in some of these protective mechanisms. Activation of p53 is an early response to genotoxic stress, and all six human IGFBP genes have predicted p53 response elements in their promoter and/or intronic regions, at least some of which are functional. IGFBP3 has been extensively characterized as a p53-inducible gene, but in some cases it is suppressed by mutant p53 forms. DNA double-strand breaks (DSBs), induced by radiotherapy and some chemotherapies, potentially lead to apoptotic cell death, senescence, or repair and recovery. DSB damage can be repaired by homologous recombination or non-homologous end-joining (NHEJ), depending on the cell cycle stage, availability of key repair proteins, and other factors. The epidermal growth factor receptor (EGFR) has been implicated in the NHEJ pathway, and EGFR inhibition may inhibit repair, promoting apoptosis and thus improving sensitivity to chemotherapy or radiotherapy. Both IGFBP-3 and IGFBP-6 interact with components of the NHEJ pathway, and IGFBP-3 can facilitate this process through direct interaction with both EGFR and the catalytic subunit of DNA-PK. Cell fate after DNA damage may in part be regulated by the balance between the sphingolipids ceramide and sphingosine-1-phosphate, and IGFBPs can influence the production of both lipids. A better understanding of the involvement of IGFBPs in the DNA damage response in cancer cells may lead to improved methods of sensitizing cancers to DNA-damaging therapies.     

Defective signal joint recombination in fanconi anemia fibroblasts reveals a role for Rad50 in V(D)J recombination

V(D)J recombination of immunoglobulin loci is dependent on the immune cell-specific Rag1 and Rag2 proteins as well as a number of ubiquitously expressed cellular DNA repair proteins that catalyze non-homologous end-joining of DNA double-strand breaks. The evolutionarily conserved Rad50/Mre11/Nibrin protein complex has a role in DNA double-strand break-repair, suggesting that these proteins, too, may participate in V(D)J recombination. Recent findings demonstrating that Rad50 function is defective in cells from patients afflicted with Fanconi anemia provide a possible mechanistic explanation for previous findings that lymphoblasts derived from these patients exhibit subtle defects in V(D)J recombination of extrachromosomal plasmid molecules. Here, we describe a series of findings that provide convincing evidence for a role of the Rad50 protein complex in V(D)J recombination. We found that the fidelity of V(D)J signal joint recombination in fibroblasts from patients afflicted with Fanconi anemia was reduced by nearly tenfold, compared to that observed in fibroblasts from normal donors. Second, we observed that antibody-mediated inhibition of the Rad50, Mre11, or Nibrin proteins reduced the fidelity of signal joint recombination significantly in wild-type cells. The latter finding was somewhat unexpected, because signal joint rejoining in cells from patients with Nijmegen breakage syndrome, which results from mutations in the Nibrin gene, occurs with normal fidelity. However, introduction of anti-Nibrin antibodies into these cells reduced the fidelity of signal joint recombination dramatically. These data reveal for the first time a role for the Rad50 complex in V(D)J recombination, and demonstrate that the protein product of the disease-causing allele responsible for Nijmegen breakage syndrome encodes a protein with residual DNA double-strand break repair activity.     

Mutation spectrum of Drosophila CNVs revealed by breakpoint sequencing

Background

The detailed study of breakpoints associated with copy number variants (CNVs) can elucidate the mutational mechanisms that generate them and the comparison of breakpoints across species can highlight differences in genomic architecture that may lead to lineage-specific differences in patterns of CNVs. Here, we provide a detailed analysis of Drosophila CNV breakpoints and contrast it with similar analyses recently carried out for the human genome.

Results

By applying split-read methods to a total of 10x coverage of 454 shotgun sequence across nine lines of D. melanogaster and by re-examining a previously published dataset of CNVs detected using tiling arrays, we identified the precise breakpoints of more than 600 insertions, deletions, and duplications. Contrasting these CNVs with those found in humans showed that in both taxa CNV breakpoints fall into three classes: blunt breakpoints; simple breakpoints associated with microhomology; and breakpoints with additional nucleotides inserted/deleted and no microhomology. In both taxa CNV breakpoints are enriched with non-B DNA sequence structures, which may impair DNA replication and/or repair. However, in contrast to human genomes, non-allelic homologous-recombination (NAHR) plays a negligible role in CNV formation in Drosophila. In flies, non-homologous repair mechanisms are responsible for simple, recurrent, and complex CNVs, including insertions of de novo sequence as large as 60 bp.

Conclusions

Humans and Drosophila differ considerably in the importance of homology-based mechanisms for the formation of CNVs, likely as a consequence of the differences in the abundance and distribution of both segmental duplications and transposable elements between the two genomes.