Nonhomologous tails direct heteroduplex rejection and mismatch correction during single-strand annealing in Saccharomyces cerevisiae.

Single-strand annealing (SSA) is initiated when a double strand break (DSB) occurs between two flanking repeated sequences, resulting in a deletion that leaves a single copy of the repeat. We studied budding yeast strains carrying two 200-bp URA3 sequences separated by 2.6 kb of spacer DNA (phage lambda) in which a site-specific DSB can be created by HO or Cas9 endonucleases. Repeat-mediated deletion requires removal of long 3'-ended single-stranded tails (flaps) by Rad1-Rad10 with the assistance of Msh2-Msh3, Saw1 and Slx4. A natural 3% divergence of unequally spaced heterologies between these repeats (designated F and A) causes a significant reduction in the frequency of SSA repair. This decrease is caused by heteroduplex rejection in which mismatches (MMs) in the annealed intermediate are recognized by the MutS (Msh2 and Msh6) components of the MM repair (MMR) pathway coupled to unwinding of the duplex by the Sgs1-Rmi1-Top3 helicase. MutL homologs, Mlh1-Pms1 (MutL), are not required for rejection but play their expected role in mismatch correction. Remarkably, heteroduplex rejection is very low in strains where the divergent repeats were immediately adjacent (Tailless strains) and the DSB was induced by Cas9. These results suggest that the presence of nonhomologous tails strongly stimulates heteroduplex rejection in SSA. DNA sequencing analysis of SSA products from the FA Tailed strain showed a gradient of correction favoring the sequence opposite each 3' end of the annealed strand. Mismatches located in the center of the repair intermediate were corrected by Msh2-Msh6 mediated mismatch correction, while correction of MMs at the extremity of the SSA intermediate often appears to use a different mechanism, possibly by 3' nonhomologous tail removal that includes part of the homologous sequence. In contrast, in FA Tailless strains there was a uniform repair of the MMs across the repeat. A distinctive pattern of correction was found in the absence of MSH2, in both Tailed and Tailless strains, different from the spectrum seen in a msh3Δ msh6Δ double mutant. Previous work has shown that SSA is Rad51-independent but dependent on the strand annealing activity of Rad52. However Rad52 becomes dispensable in a Tailless construct where the DSB is induced by Cas9 or in transformation of a plasmid where SSA occurs in the absence of nonhomologous tails.

Unequal redundancy in maize knotted1 homeobox genes.

The knotted1 (kn1) homeobox (knox) gene family was first identified through gain-of-function dominant mutants in maize (Zea mays). Class I knox members are expressed in meristems but excluded from leaves. In maize, a loss-of-function phenotype has only been characterized for kn1. To assess the function of another knox member, we characterized a loss-of-function mutation of rough sheath1 (rs1). rs1-mum1 has no phenotype alone but exacerbates several aspects of the kn1 phenotype. In permissive backgrounds in which kn1 mutants grow to maturity, loss of a single copy of rs1 enhances the tassel branch reduction phenotype, while loss of both copies results in limited shoots. In less introgressed lines, double mutants can grow to maturity but are shorter. Using a KNOX antibody, we demonstrate that RS1 binds in vivo to some of the KN1 target genes, which could partially explain why KN1 binds many genes but modulates few. Our results demonstrate an unequal redundancy between knox genes, with a role for rs1 only revealed in the complete absence of kn1.

Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP regulates CPC-spindle interaction to ensure proper microtubule dynamics.

Dynamic microtubules facilitate chromosome arrangement before anaphase, whereas during anaphase microtubule stability assists chromosome separation. Changes in microtubule dynamics at the metaphase-anaphase transition are regulated by Cdk1. Cdk1-mediated phosphorylation of Sli15/INCENP promotes preanaphase microtubule dynamics by preventing chromosomal passenger complex (CPC; Sli15/INCENP, Bir1/Survivin, Nbl1/Borealin, Ipl1/Aurora) association with spindles. However, whether Cdk1 has sole control over microtubule dynamics, and how CPC-microtubule association influences microtubule behavior, are unclear. Here, we show that Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP modulates microtubule dynamics by preventing CPC binding to the preanaphase spindle and to the central spindle until late anaphase, facilitating spatiotemporal control of microtubule dynamics required for proper metaphase centromere positioning and anaphase spindle elongation. Decreased Ipl1-dependent Sli15 phosphorylation drives direct CPC binding to microtubules, revealing how the CPC influences microtubule dynamics. We propose that Cdk1 and Ipl1/Aurora cooperatively modulate microtubule dynamics and that Ipl1/Aurora-dependent phosphorylation of Sli15 controls spindle function by excluding the CPC from spindle regions engaged in microtubule polymerization.

Overlapping kinetochore targets of CK2 and Aurora B kinases in mitotic regulation.

Protein kinase CK2 is one of the most conserved kinases in eukaryotic cells and plays essential roles in diverse processes. While we know that CK2 plays a role(s) in cell division, our understanding of how CK2 regulates cell cycle progression is limited. In this study, we revealed a regulatory role for CK2 in kinetochore function. The kinetochore is a multi-protein complex that assembles on the centromere of a chromosome and functions to attach chromosomes to spindle microtubules. To faithfully segregate chromosomes and maintain genomic integrity, the kinetochore is tightly regulated by multiple mechanisms, including phosphorylation by Aurora B kinase. We found that a loss of CK2 kinase activity inhibits anaphase spindle elongation and results in chromosome missegregation. Moreover, a lack of CK2 activates the spindle assembly checkpoint. We demonstrate that CK2 associates with Mif2, the Saccharomyces cerevisiae homologue of human CENP-C, which serves as an important link between the inner and outer kinetochore. Furthermore, we show Mif2 and the inner kinetochore protein Ndc10 are phosphorylated by CK2, and this phosphorylation plays antagonistic and synergistic roles with Aurora B phosphorylation of these targets, respectively.

Nbl1p: a Borealin/Dasra/CSC-1-like protein essential for Aurora/Ipl1 complex function and integrity in Saccharomyces cerevisiae.

The Aurora kinase complex, also called the chromosomal passenger complex (CPC), is essential for faithful chromosome segregation and completion of cell division. In Fungi and Animalia, this complex consists of the kinase Aurora B/AIR-2/Ipl1p, INCENP/ICP-1/Sli15p, and Survivin/BIR-1/Bir1p. A fourth subunit, Borealin/Dasra/CSC-1, is required for CPC targeting to centromeres and central spindles and has only been found in Animalia. Here we identified a new core component of the CPC in budding yeast, Nbl1p. NBL1 is essential for viability and nbl1 mutations cause chromosome missegregation and lagging chromosomes. Nbl1p colocalizes and copurifies with the CPC, and it is essential for CPC localization, stability, integrity, and function. Nbl1p is related to the N-terminus of Borealin/Dasra/CSC-1 and is similarly involved in connecting the other CPC subunits. Distant homology searching identified nearly 200, mostly unannotated, Borealin/Dasra/CSC-1-related proteins from nearly 150 species within Fungi and Animalia. Analysis of the sequence of these proteins, combined with comparative protein structure modeling of Bir1p-Nbl1p-Sli15p using the crystal structure of the human Survivin-Borealin-INCENP complex, revealed a striking structural conservation across a broad range of species. Our biological and computational analyses therefore establish that the fundamental design of the CPC is conserved from Fungi to Animalia.