Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination

Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ;6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids-1

<p><b>Copyright information:</b></p><p>Taken from "A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids"</p><p>http://www.biomedcentral.com/1471-2199/9/54</p><p>BMC Molecular Biology 2008;9():54-54.</p><p>Published online 4 Jun 2008</p><p>PMCID:PMC2443376.</p><p></p

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids-7

<p><b>Copyright information:</b></p><p>Taken from "A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids"</p><p>http://www.biomedcentral.com/1471-2199/9/54</p><p>BMC Molecular Biology 2008;9():54-54.</p><p>Published online 4 Jun 2008</p><p>PMCID:PMC2443376.</p><p></p

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids-0

E second cycle of amplification. (D) The third cycle of amplification and fluorescent signal generation; (E), (Fa) and (Fb), The fourth cycle of amplification and fluorescent signal generation.<p><b>Copyright information:</b></p><p>Taken from "A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids"</p><p>http://www.biomedcentral.com/1471-2199/9/54</p><p>BMC Molecular Biology 2008;9():54-54.</p><p>Published online 4 Jun 2008</p><p>PMCID:PMC2443376.</p><p></p

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids-2

Nsgenic soybean ranging from 0.01 to 100 ng with CaMV35s promoter real-time PCR system; b: Initial DNA concentration versus Ct standard curve; (B) The standard curve of NOS terminator quantification. a: Amplification plot generated by five dilutions (10-fold) of transgenic soybean ranging from 0.01 to 100 ng with NOS terminator real-time PCR system; b: Initial DNA concentration versus Ct standard curve.<p><b>Copyright information:</b></p><p>Taken from "A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids"</p><p>http://www.biomedcentral.com/1471-2199/9/54</p><p>BMC Molecular Biology 2008;9():54-54.</p><p>Published online 4 Jun 2008</p><p>PMCID:PMC2443376.</p><p></p

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids-6

E second cycle of amplification. (D) The third cycle of amplification and fluorescent signal generation; (E), (Fa) and (Fb), The fourth cycle of amplification and fluorescent signal generation.<p><b>Copyright information:</b></p><p>Taken from "A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids"</p><p>http://www.biomedcentral.com/1471-2199/9/54</p><p>BMC Molecular Biology 2008;9():54-54.</p><p>Published online 4 Jun 2008</p><p>PMCID:PMC2443376.</p><p></p

The replanted main shoot and tiller of <i>dwt1</i> reproduce the main-shoot-dominance phenotype.

<p>A. Morphology of mature plants developed from replanted main shoot of the wild type (RPwM), replanted tillers of the wild type (RPwT), replanted main shoot of <i>dwt1</i> (RPdM) and replanted tillers of <i>dwt1</i> (RPdT). Bar = 5 cm. B. The culm length of both main shoots (MS) and tillers (TS) of replanted plants. Culm length of 15 main shoots and 60 tillers of wild-type plants, 15 main shoots and 55 tillers of mutant plants were measured at mature stage. Error bars indicate SD, and the very significant differences from the wild type are marked (**p<0.01, Student's <i>t</i> test).</p

<i>dwt1</i> has defects in cell elongation and cell proliferation.

<p>A. The second internode of mature wild-type (left) and <i>dwt1</i> (right) plants. Bar = 1 cm. B. The length of second internode at mature stage. n = 30. Error bars indicate SD, and the very significant differences from wild type are marked (**p<0.01, Student's <i>t</i> test). C and D. Longitudinal sections through the middle of the second internode of the wild type (C) and <i>dwt1</i> (D) after the heading stage. Bar = 100 µm. E. Longitudinal and transverse length of the cells in the second internode. 12 second internodes from 3 individual mature plants were used, and 10 cells of each internode were measured. Error bars indicate SD, and the very significant differences from wild type are marked (**p<0.01, Student's <i>t</i> test). F. Cell number of the whole second internodes in mature plants. n = 5. Internode sample harvested from 5 individual plants were used for section. Error bars indicate SD, and the very significant differences from wild type are marked (**p<0.01, Student's <i>t</i> test).</p

The <i>dwt1</i> mutant plants display morphological defects.

<p>A. Morphology of the wild type and <i>dwt1</i> plants after heading. Bar = 10 cm. B. Mature main shoots with leaves removed from the culm. Arrowheads point to the nodes. Bar = 10 cm. C. Mature tillers with leaves removed from the culm. Arrowheads point to the nodes. Bar = 10 cm. D. Close-up view of the internodes after heading stage. From left to right: the 2^nd node of the wild type, the 2^nd internode, the 2^nd and 3^rd internodes, the 2^nd, 3^rd, and 4^th internodes of <i>dwt1</i> mutant. Bar = 0.5 cm. E. Frequency of normal and short internodes in wild-type and <i>dwt1</i> main shoot (MS) and tiller shoot (TS). 2^nd: only 2^nd internode short; 2^nd, 3^rd: both 2^nd and 3^rd internodes short; 2^nd, 3^rd, 4^th: all 2^nd, 3^rd, 4^th internodes short. Elongation pattern of main shoots and tillers of both wild type and <i>dwt1</i> mutants were evaluated at mature stage, and 25 main shoots and 100 tillers of wild type and 25 main shoots and 143 tillers of mutant plants were observed. F. Morphology of panicles from the main shoot (MS) and tillers (TS) of wild type (WT) and <i>dwt1</i> after heading. Bar = 2 cm.</p

Molecular characterization of <i>DWT1</i>.

<p>A. Map-based cloning of the <i>DWT1</i> gene. The chromosomal region containing <i>DWT1</i> is diagrammed as the top line with molecular markers shown above the line. The number below the corresponding markers indicates the numbers of recombinants between the markers and <i>DWT1</i>. The BAC clones are shown as overlapping lines. B. Structure of the <i>DWT1</i> gene. The mutant sequence has one nucleotide C₇₆₃ deletion in the second exon. Black boxes indicate exons, white boxes indicate UTRs and lines indicate introns. The grey box shows the homeobox domain. C. The plant stature of <i>dwt1</i>, the wild type, and complemented transgenic plants (DWT1-COM). Bar = 10 cm. D. The morphology of the second internodes of <i>dwt1</i>, wild type, and complemented transgenic plants (DWT1-COM). Bar = 2 cm. E–H. Nuclear localization of DWT1 protein. YFP fluorescence image (E), light view (F), PI stained image (G) and overlay of the three images (H) of <i>Nicotiana benthamiana</i> leaf epidermal cell transformed with the 35S:DWT1-YFP construct.</p