Limits...
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.

Show MeSH

Related in: MedlinePlus

Average number of gamma-H2AX foci per nucleus from unirradiated mushrooms at 2 hr past karyogamy and 1 hr after irradiation with 60 krad. Error bars are 95% confidence intervals. There are significantly more gamma-H2AX foci in irradiated mushrooms for each genotype (Student t test, P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3815056&req=5

fig9: Average number of gamma-H2AX foci per nucleus from unirradiated mushrooms at 2 hr past karyogamy and 1 hr after irradiation with 60 krad. Error bars are 95% confidence intervals. There are significantly more gamma-H2AX foci in irradiated mushrooms for each genotype (Student t test, P < 0.01).

Mentions: The lower number of gamma-H2AX foci in nbs1-2 prophase nuclei suggested a defect in DSB formation, more rapid repair of DSBs, or a defect in H2AX phosphorylation. In the S-phase checkpoint response and in meiosis, Nbs1 activates ATM, and ATM then performs a wide variety of functions, including phosphorylating H2AX (Burma et al. 2001). We do not predict that the nbs1-2 mutation affects binding to ATM; however, it is possible that there is some effect on ATM activation if Nbs1 cannot localize efficiently to the DSB. To ask if H2AX can be phosphorylated with wild-type kinetics in nbs1-2, we irradiated wild-type and nbs1-2 with ionizing radiation 2 hr after the beginning of meiosis and then stained nuclei with anti-gamma-H2AX. We found a significant increase in the number of gamma-H2AX foci 1 hr after irradiation in both wild-type and nbs1-2 (P < 0.01) (Figure 9). There was a similar increase in the number of foci in wild-type and nbs1-2 (62% and 73% increases relative to unirradiated, respectively), indicating that H2AX is phosphorylated in response to DSBs in nbs1-2 at a level similar to that in wild-type.


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)

Average number of gamma-H2AX foci per nucleus from unirradiated mushrooms at 2 hr past karyogamy and 1 hr after irradiation with 60 krad. Error bars are 95% confidence intervals. There are significantly more gamma-H2AX foci in irradiated mushrooms for each genotype (Student t test, P < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: Average number of gamma-H2AX foci per nucleus from unirradiated mushrooms at 2 hr past karyogamy and 1 hr after irradiation with 60 krad. Error bars are 95% confidence intervals. There are significantly more gamma-H2AX foci in irradiated mushrooms for each genotype (Student t test, P < 0.01).
Mentions: The lower number of gamma-H2AX foci in nbs1-2 prophase nuclei suggested a defect in DSB formation, more rapid repair of DSBs, or a defect in H2AX phosphorylation. In the S-phase checkpoint response and in meiosis, Nbs1 activates ATM, and ATM then performs a wide variety of functions, including phosphorylating H2AX (Burma et al. 2001). We do not predict that the nbs1-2 mutation affects binding to ATM; however, it is possible that there is some effect on ATM activation if Nbs1 cannot localize efficiently to the DSB. To ask if H2AX can be phosphorylated with wild-type kinetics in nbs1-2, we irradiated wild-type and nbs1-2 with ionizing radiation 2 hr after the beginning of meiosis and then stained nuclei with anti-gamma-H2AX. We found a significant increase in the number of gamma-H2AX foci 1 hr after irradiation in both wild-type and nbs1-2 (P < 0.01) (Figure 9). There was a similar increase in the number of foci in wild-type and nbs1-2 (62% and 73% increases relative to unirradiated, respectively), indicating that H2AX is phosphorylated in response to DSBs in nbs1-2 at a level similar to that in wild-type.

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