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DNA replication arrest leads to enhanced homologous recombination and cell death in meristems of rice OsRecQl4 mutants.

ABSTRACT: BACKGROUND: Mammalian BLM helicase is involved in DNA replication, DNA repair and homologous recombination (HR). These DNA transactions are associated tightly with cell division and are important for maintaining genome stability. However, unlike in mammals, cell division in higher plants is restricted mainly to the meristem, thus genome maintenance at the meristem is critical. The counterpart of BLM in Arabidopsis (AtRecQ4A) has been identified and its role in HR and in the response to DNA damage has been confirmed. However, the function of AtRecQ4A in the meristem during replication stress has not yet been well elucidated. RESULTS: We isolated the BLM counterpart gene OsRecQl4 from rice and analyzed its function using a reverse genetics approach. Osrecql4 mutant plants showed hypersensitivity to DNA damaging agents and enhanced frequency of HR compared to wild-type (WT) plants. We further analyzed the effect of aphidicolin--an inhibitor of S-phase progression via its inhibitory effect on DNA polymerases--on genome stability in the root meristem in osrecql4 mutant plants and corresponding WT plants. The following effects were observed upon aphidicolin treatment: a) comet assay showed induction of DNA double-strand breaks (DSBs) in mutant plants, b) TUNEL assay showed enhanced DNA breaks at the root meristem in mutant plants, c) a recombination reporter showed enhanced HR frequency in mutant calli, d) propidium iodide (PI) staining of root tips revealed an increased incidence of cell death in the meristem of mutant plants. CONCLUSIONS: These results demonstrate that the aphidicolin-sensitive phenotype of osrecql4 mutants was in part due to induced DSBs and cell death, and that OsRecQl4 plays an important role as a caretaker, maintaining genome stability during DNA replication stress in the rice meristem.

SUBMITTER: Kwon YI

PROVIDER: S-EPMC3648487 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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DNA replication arrest leads to enhanced homologous recombination and cell death in meristems of rice OsRecQl4 mutants.

Kwon Yong-Ik YI Abe Kiyomi K Endo Masaki M Osakabe Keishi K Ohtsuki Namie N Nishizawa-Yokoi Ayako A Tagiri Akemi A Saika Hiroaki H Toki Seiichi S

BMC plant biology 20130412

<h4>Background</h4>Mammalian BLM helicase is involved in DNA replication, DNA repair and homologous recombination (HR). These DNA transactions are associated tightly with cell division and are important for maintaining genome stability. However, unlike in mammals, cell division in higher plants is restricted mainly to the meristem, thus genome maintenance at the meristem is critical. The counterpart of BLM in Arabidopsis (AtRecQ4A) has been identified and its role in HR and in the response to DN ...[more]

PMID: 23586618

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Project description:The Saccharomyces cerevisae RAD3 gene is the homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Some mutant alleles of RAD3 (rad3-101 and rad3-102) have partial defects in DNA repair and a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. The systems previously used to characterize the hyper-Rec phenotype of rad3 strains do not detect the reciprocal products of mitotic recombination. We have further characterized these events using a system in which the reciprocal products of mitotic recombination are recovered. Both rad3-101 and rad3-102 elevate the frequency of reciprocal crossovers about 100-fold. Mapping of these events shows that three-quarters of these crossovers reflect DSBs formed at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. We mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister-chromatid recombination, are a major source of mitotic recombination between homologous chromosomes.

Project description:The Saccharomyces cerevisae RAD3 gene is homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER) and transcription. Mutant alleles of RAD3 have been identified (rad3-101 and rad3-102) that have partial defects in DNA repair associated with a strong hyper-recombination (hyper-Rec) phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs) by replication. We have further characterized these events using a system in which the reciprocal products of mitotic recombination between homologs are recovered as red and white sectored colonies. Both rad3-101 and rad3-102 elevate the frequency of sectored colonies about 100-fold. Subsequent mapping of these events shows that three-quarters of crossovers between homologs induced in hyper-Rec rad3 mutants reflect DSBs formed in at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs). The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs). The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. In addition to examining crossovers on chromosome V, we mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister-chromatid recombination, are a major source of mitotic recombination between homologous chromosomes.

Dataset Information

DNA replication arrest leads to enhanced homologous recombination and cell death in meristems of rice OsRecQl4 mutants.

Publications

DNA replication arrest leads to enhanced homologous recombination and cell death in meristems of rice OsRecQl4 mutants.

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