SUMO fosters assembly and functionality of the MutSγ complex to facilitate meiotic crossing over.

Crossing over is essential for chromosome segregation during meiosis. Protein modification by SUMO is implicated in crossover control, but pertinent targets have remained elusive. Here we identify Msh4 as a target of SUMO-mediated crossover regulation. Msh4 and Msh5 constitute the MutSγ complex, which stabilizes joint-molecule (JM) recombination intermediates and facilitates their resolution into crossovers. Msh4 SUMOylation enhances these processes to ensure that each chromosome pair acquires at least one crossover. Msh4 is directly targeted by E2 conjugase Ubc9, initially becoming mono-SUMOylated in response to DNA double-strand breaks, then multi/poly-SUMOylated forms arise as homologs fully engage. Mechanistically, SUMOylation fosters interaction between Msh4 and Msh5. We infer that initial SUMOylation of Msh4 enhances assembly of MutSγ in anticipation of JM formation, while secondary SUMOylation may promote downstream functions. Regulation of Msh4 by SUMO is distinct and independent of its previously described stabilization by phosphorylation, defining MutSγ as a hub for crossover control.

PCNA activates the MutLγ endonuclease to promote meiotic crossing over.

During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation1. Defective crossing over causes infertility, miscarriage and congenital disease. Each pair of chromosomes attains at least one crossover via the formation and biased resolution of recombination intermediates known as double Holliday junctions2,3. A central principle of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands4. The endonuclease activity of the MutLγ complex has been implicated in the resolution of crossovers5-10, but the mechanisms that activate and direct strand-specific cleavage remain unknown. Here we show that the sliding clamp PCNA is important for crossover-biased resolution. In vitro assays with human enzymes show that PCNA and its loader RFC are sufficient to activate the MutLγ endonuclease. MutLγ is further stimulated by a co-dependent activity of the pro-crossover factors EXO1 and MutSγ, the latter of which binds Holliday junctions11. MutLγ also binds various branched DNAs, including Holliday junctions, but does not show canonical resolvase activity, implying that the endonuclease incises adjacent to junction branch points to achieve resolution. In vivo, RFC facilitates MutLγ-dependent crossing over in budding yeast. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover resolution and the initiation steps of DNA mismatch repair12,13 and evoke a novel model for crossover-specific resolution of double Holliday junctions during meiosis.

Proline-rich protein PRR19 functions with cyclin-like CNTD1 to promote meiotic crossing over in mouse.

Orderly chromosome segregation is enabled by crossovers between homologous chromosomes in the first meiotic division. Crossovers arise from recombination-mediated repair of programmed DNA double-strand breaks (DSBs). Multiple DSBs initiate recombination, and most are repaired without crossover formation, although one or more generate crossovers on each chromosome. Although the underlying mechanisms are ill-defined, the differentiation and maturation of crossover-specific recombination intermediates requires the cyclin-like CNTD1. Here, we identify PRR19 as a partner of CNTD1. We find that, like CNTD1, PRR19 is required for timely DSB repair and the formation of crossover-specific recombination complexes. PRR19 and CNTD1 co-localise at crossover sites, physically interact, and are interdependent for accumulation, indicating a PRR19-CNTD1 partnership in crossing over. Further, we show that CNTD1 interacts with a cyclin-dependent kinase, CDK2, which also accumulates in crossover-specific recombination complexes. Thus, the PRR19-CNTD1 complex may enable crossover differentiation by regulating CDK2.

Regulated Proteolysis of MutSγ Controls Meiotic Crossing Over.

Crossover recombination is essential for accurate chromosome segregation during meiosis. The MutSγ complex, Msh4-Msh5, facilitates crossing over by binding and stabilizing nascent recombination intermediates. We show that these activities are governed by regulated proteolysis. MutSγ is initially inactive for crossing over due to an N-terminal degron on Msh4 that renders it unstable by directly targeting proteasomal degradation. Activation of MutSγ requires the Dbf4-dependent kinase Cdc7 (DDK), which directly phosphorylates and thereby neutralizes the Msh4 degron. Genetic requirements for Msh4 phosphorylation indicate that DDK targets MutSγ only after it has bound to nascent joint molecules (JMs) in the context of synapsing chromosomes. Overexpression studies confirm that the steady-state level of Msh4, not phosphorylation per se, is the critical determinant for crossing over. At the DNA level, Msh4 phosphorylation enables the formation and crossover-biased resolution of double-Holliday Junction intermediates. Our study establishes regulated protein degradation as a fundamental mechanism underlying meiotic crossing over.

Tetramerization at Low pH Licenses DNA Methylation Activity of M.HpyAXI in the Presence of Acid Stress.

Methylation of genomic DNA can influence the transcription profile of an organism and may generate phenotypic diversity for rapid adaptation in a dynamic environment. M.HpyAXI is a Type III DNA methyltransferase present in Helicobacter pylori and is upregulated at low pH. This enzyme may alter the expression of critical genes to ensure the survival of this pathogen at low pH inside the human stomach. M.HpyAXI methylates the adenine in the target sequence (5'-GCAG-3') and shows maximal activity at pH 5.5. Type III DNA methyltransferases are found to form an inverted dimer in the functional form. We observe that M.HpyAXI forms a nonfunctional dimer at pH 8.0 that is incapable of DNA binding and methylation activity. However, at pH 5.5, two such dimers associate to form a tetramer that now includes two functional dimers that can bind and methylate the target DNA sequence. Overall, we observe that the pH-dependent tetramerization of M.HpyAXI ensures that the enzyme is licensed to act only in the presence of acid stress.

Mechanism of formation of a toroid around DNA by the mismatch sensor protein.

The DNA mismatch repair (MMR) pathway removes errors that appear during genome replication. MutS is the primary mismatch sensor and forms an asymmetric dimer that encircles DNA to bend it to scan for mismatches. The mechanism utilized to load DNA into the central tunnel was unknown and the origin of the force required to bend DNA was unclear. We show that, in absence of DNA, MutS forms a symmetric dimer wherein a gap exists between the monomers through which DNA can enter the central tunnel. The comparison with structures of MutS-DNA complexes suggests that the mismatch scanning monomer (Bm) will move by nearly 50 Å to associate with the other monomer (Am). Consequently, the N-terminal domains of both monomers will press onto DNA to bend it. The proposed mechanism of toroid formation evinces that the force required to bend DNA arises primarily due to the movement of Bm and hence, the MutS dimer acts like a pair of pliers to bend DNA. We also shed light on the allosteric mechanism that influences the expulsion of adenosine triphosphate from Am on DNA binding. Overall, this study provides mechanistic insight regarding the primary event in MMR i.e. the assembly of the MutS-DNA complex.