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A mutation in the FHA domain of Coprinus cinereus Nbs1 Leads to Spo11-independent meiotic recombination and chromosome segregation.

Crown KN, Savytskyy OP, Malik SB, Logsdon J, Williams RS, Tainer JA, Zolan ME - G3 (Bethesda) (2013)

Bottom Line: We propose that replication-dependent DSBs, resulting from defective replication fork protection and processing by the Mre11-Rad50-Nbs1 complex, are competent to form meiotic crossovers in C. cinereus, and that these crossovers lead to high levels of faithful chromosome segregation.In addition, although crossover distribution is altered in nbs1-2, the majority of crossovers were found in subtelomeric regions, as in wild-type.Therefore, the location of crossovers in C. cinereus is maintained when DSBs are induced via a Spo11-independent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University, Bloomington, Indiana 47405.

ABSTRACT
Nbs1, a core component of the Mre11-Rad50-Nbs1 complex, plays an essential role in the cellular response to DNA double-strand breaks (DSBs) and poorly understood roles in meiosis. We used the basidiomycete Coprinus cinereus to examine the meiotic roles of Nbs1. We identified the C. cinereus nbs1 gene and demonstrated that it corresponds to a complementation group previously known as rad3. One allele, nbs1-2, harbors a point mutation in the Nbs1 FHA domain and has a mild spore viability defect, increased frequency of meiosis I nondisjunction, and an altered crossover distribution. The nbs1-2 strain enters meiosis with increased levels of phosphorylated H2AX, which we hypothesize represent unrepaired DSBs formed during premeiotic replication. In nbs1-2, there is no apparent induction of Spo11-dependent DSBs during prophase. We propose that replication-dependent DSBs, resulting from defective replication fork protection and processing by the Mre11-Rad50-Nbs1 complex, are competent to form meiotic crossovers in C. cinereus, and that these crossovers lead to high levels of faithful chromosome segregation. In addition, although crossover distribution is altered in nbs1-2, the majority of crossovers were found in subtelomeric regions, as in wild-type. Therefore, the location of crossovers in C. cinereus is maintained when DSBs are induced via a Spo11-independent mechanism.

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Anti-gamma-H2AX localization on wild-type meiotic chromosome spreads. Images in the left column are DAPI-stained chromosome spreads; the stage of prophase is indicated on the image. Images in the middle column are of anti-gamma-H2AX counterstained with a TRITC-conjugated secondary antibody, and images in the right column are the color-combined images. Prefusion, n = 42; leptotene, n = 47; zygotene, n = 50; pachytene, n = 48; diplotene, n = 60. Scale bars represent 2 µm.
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fig6: Anti-gamma-H2AX localization on wild-type meiotic chromosome spreads. Images in the left column are DAPI-stained chromosome spreads; the stage of prophase is indicated on the image. Images in the middle column are of anti-gamma-H2AX counterstained with a TRITC-conjugated secondary antibody, and images in the right column are the color-combined images. Prefusion, n = 42; leptotene, n = 47; zygotene, n = 50; pachytene, n = 48; diplotene, n = 60. Scale bars represent 2 µm.

Mentions: A time course of wild-type meiotic chromosome spreads was stained with anti-gamma-H2AX to characterize wild-type DSB formation and repair during prophase I (Figure 6 and Figure 8). In prefusion (n = 42) and fusion nuclei (n = 13), there were low levels of gamma-H2AX staining; these foci likely represent a combination of background staining and DSBs formed during premeiotic replication. At leptotene (n = 47) and zygotene (n = 50), there was a dramatic induction of H2AX phosphorylation that remained through the end of pachytene (n = 48), disappearing during diffuse diplotene (n = 60) to levels lower than those in prefusion nuclei.


A mutation in the FHA domain of Coprinus cinereus Nbs1 Leads to Spo11-independent meiotic recombination and chromosome segregation.

Crown KN, Savytskyy OP, Malik SB, Logsdon J, Williams RS, Tainer JA, Zolan ME - G3 (Bethesda) (2013)

Anti-gamma-H2AX localization on wild-type meiotic chromosome spreads. Images in the left column are DAPI-stained chromosome spreads; the stage of prophase is indicated on the image. Images in the middle column are of anti-gamma-H2AX counterstained with a TRITC-conjugated secondary antibody, and images in the right column are the color-combined images. Prefusion, n = 42; leptotene, n = 47; zygotene, n = 50; pachytene, n = 48; diplotene, n = 60. Scale bars represent 2 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3815056&req=5

fig6: Anti-gamma-H2AX localization on wild-type meiotic chromosome spreads. Images in the left column are DAPI-stained chromosome spreads; the stage of prophase is indicated on the image. Images in the middle column are of anti-gamma-H2AX counterstained with a TRITC-conjugated secondary antibody, and images in the right column are the color-combined images. Prefusion, n = 42; leptotene, n = 47; zygotene, n = 50; pachytene, n = 48; diplotene, n = 60. Scale bars represent 2 µm.
Mentions: A time course of wild-type meiotic chromosome spreads was stained with anti-gamma-H2AX to characterize wild-type DSB formation and repair during prophase I (Figure 6 and Figure 8). In prefusion (n = 42) and fusion nuclei (n = 13), there were low levels of gamma-H2AX staining; these foci likely represent a combination of background staining and DSBs formed during premeiotic replication. At leptotene (n = 47) and zygotene (n = 50), there was a dramatic induction of H2AX phosphorylation that remained through the end of pachytene (n = 48), disappearing during diffuse diplotene (n = 60) to levels lower than those in prefusion nuclei.

Bottom Line: We propose that replication-dependent DSBs, resulting from defective replication fork protection and processing by the Mre11-Rad50-Nbs1 complex, are competent to form meiotic crossovers in C. cinereus, and that these crossovers lead to high levels of faithful chromosome segregation.In addition, although crossover distribution is altered in nbs1-2, the majority of crossovers were found in subtelomeric regions, as in wild-type.Therefore, the location of crossovers in C. cinereus is maintained when DSBs are induced via a Spo11-independent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Indiana University, Bloomington, Indiana 47405.

ABSTRACT
Nbs1, a core component of the Mre11-Rad50-Nbs1 complex, plays an essential role in the cellular response to DNA double-strand breaks (DSBs) and poorly understood roles in meiosis. We used the basidiomycete Coprinus cinereus to examine the meiotic roles of Nbs1. We identified the C. cinereus nbs1 gene and demonstrated that it corresponds to a complementation group previously known as rad3. One allele, nbs1-2, harbors a point mutation in the Nbs1 FHA domain and has a mild spore viability defect, increased frequency of meiosis I nondisjunction, and an altered crossover distribution. The nbs1-2 strain enters meiosis with increased levels of phosphorylated H2AX, which we hypothesize represent unrepaired DSBs formed during premeiotic replication. In nbs1-2, there is no apparent induction of Spo11-dependent DSBs during prophase. We propose that replication-dependent DSBs, resulting from defective replication fork protection and processing by the Mre11-Rad50-Nbs1 complex, are competent to form meiotic crossovers in C. cinereus, and that these crossovers lead to high levels of faithful chromosome segregation. In addition, although crossover distribution is altered in nbs1-2, the majority of crossovers were found in subtelomeric regions, as in wild-type. Therefore, the location of crossovers in C. cinereus is maintained when DSBs are induced via a Spo11-independent mechanism.

Show MeSH
Related in: MedlinePlus