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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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The template-based homology modeling program PHYRE was used to predict the location of functional domains within C. cinereus Nbs1. Numbers represent the amino acid boundaries of the functional domains. Locations of the predicted protein changes caused by nbs1 mutations are represented by ↓. The nbs1-2 mutation leads to an amino acid substitution, and nbs1-1, nbs1-3, and nbs1-5 cause truncations. For nbs1-1, only the truncation at amino acid 406 is shown.
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fig2: The template-based homology modeling program PHYRE was used to predict the location of functional domains within C. cinereus Nbs1. Numbers represent the amino acid boundaries of the functional domains. Locations of the predicted protein changes caused by nbs1 mutations are represented by ↓. The nbs1-2 mutation leads to an amino acid substitution, and nbs1-1, nbs1-3, and nbs1-5 cause truncations. For nbs1-1, only the truncation at amino acid 406 is shown.

Mentions: Using the predicted gene sequence to design primers, we amplified and sequenced nbs1 from DNA and cDNA of a wild-type strain. C. cinereus nbs1 is 3528 bp long and has 10 inferred introns, and the coding region is 3000 bp. The predicted protein has 999 amino acids and a predicted molecular weight of 110 kD (Figure 2). Using the template-based homology-modeling program PHYRE (Kelley and Sternberg 2009), we found that C. cinereus Nbs1 has the three conserved protein domains found in all other Nbs1 orthologs: an FHA domain and two tandem BRCT domains (Figure 2, Figure S1, and Figure S2). Most of the amino acids that form the binding pocket of the FHA domain, those that comprise the FHA/BRCT1 interface, and those that contact phosphorylated residues are precisely conserved or have conservative changes (Figure S1 and Figure S2). The C-terminus of the protein is predicted to be disordered, although a BLAST alignment between human Nbs1 and C. cinereus Nbs1 shows that the Mre11-binding peptide motif and the 100 amino acids surrounding it are highly conserved in C. cinereus (Figure S3). This region has 25% sequence identity and 40% similarity with human Nbs1. The exact Mre11 binding motif (NFKKFKK) is almost 100% identical to that of the human protein, except the last lysine is substituted with arginine. We were unable to identify an ATM-binding motif.


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)

The template-based homology modeling program PHYRE was used to predict the location of functional domains within C. cinereus Nbs1. Numbers represent the amino acid boundaries of the functional domains. Locations of the predicted protein changes caused by nbs1 mutations are represented by ↓. The nbs1-2 mutation leads to an amino acid substitution, and nbs1-1, nbs1-3, and nbs1-5 cause truncations. For nbs1-1, only the truncation at amino acid 406 is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: The template-based homology modeling program PHYRE was used to predict the location of functional domains within C. cinereus Nbs1. Numbers represent the amino acid boundaries of the functional domains. Locations of the predicted protein changes caused by nbs1 mutations are represented by ↓. The nbs1-2 mutation leads to an amino acid substitution, and nbs1-1, nbs1-3, and nbs1-5 cause truncations. For nbs1-1, only the truncation at amino acid 406 is shown.
Mentions: Using the predicted gene sequence to design primers, we amplified and sequenced nbs1 from DNA and cDNA of a wild-type strain. C. cinereus nbs1 is 3528 bp long and has 10 inferred introns, and the coding region is 3000 bp. The predicted protein has 999 amino acids and a predicted molecular weight of 110 kD (Figure 2). Using the template-based homology-modeling program PHYRE (Kelley and Sternberg 2009), we found that C. cinereus Nbs1 has the three conserved protein domains found in all other Nbs1 orthologs: an FHA domain and two tandem BRCT domains (Figure 2, Figure S1, and Figure S2). Most of the amino acids that form the binding pocket of the FHA domain, those that comprise the FHA/BRCT1 interface, and those that contact phosphorylated residues are precisely conserved or have conservative changes (Figure S1 and Figure S2). The C-terminus of the protein is predicted to be disordered, although a BLAST alignment between human Nbs1 and C. cinereus Nbs1 shows that the Mre11-binding peptide motif and the 100 amino acids surrounding it are highly conserved in C. cinereus (Figure S3). This region has 25% sequence identity and 40% similarity with human Nbs1. The exact Mre11 binding motif (NFKKFKK) is almost 100% identical to that of the human protein, except the last lysine is substituted with arginine. We were unable to identify an ATM-binding motif.

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