Both distance and flowering synchrony influenced pollination patterns in E. angustifolia. Our results suggest that pollen movement between incompatible mates and flowering asynchrony could be contributing to the reduced seed set observed in small E. angustifolia remnants. However, we also found that individual plants receive pollen from a diverse group of pollen donors.

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During meiosis, homologous recombination repairs programmed DNA double-stranded breaks. Meiotic recombination physically links the homologous chromosomes (“homologs”), creating the tension between them that is required for their segregation. The central recombinase in this process is Dmc1. Dmc1’s activity is regulated by its accessory factors including the heterodimeric protein Mei5-Sae3 and Rad51. We use a gain-of-function dmc1 mutant, dmc1-E157D, that bypasses Mei5-Sae3 to gain insight into the role of this accessory factor and its relationship to mitotic recombinase Rad51, which also functions as a Dmc1 accessory protein during meiosis. We find that Mei5-Sae3 has a role in filament formation and stability, but not in the bias of recombination partner choice that favors homolog over sister chromatids. Analysis of meiotic recombination intermediates suggests that Mei5-Sae3 and Rad51 function independently in promoting filament stability. In spite of its ability to load onto single-stranded DNA and carry out recombination in the absence of Mei5-Sae3, recombination promoted by the Dmc1 mutant is abnormal in that it forms foci in the absence of DNA breaks, displays unusually high levels of multi-chromatid and intersister joint molecule intermediates, as well as high levels of ectopic recombination products. We use super-resolution microscopy to show that the mutant protein forms longer foci than those formed by wild-type Dmc1. Our data support a model in which longer filaments are more prone to engage in aberrant recombination events, suggesting that filament lengths are normally limited by a regulatory mechanism that functions to prevent recombination-mediated genome rearrangements.

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, the two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway. INTRODUCTION:Homologous recombination (HR) is a high-fidelity mechanism of repair of DNA double-stranded breaks (DSBs), inter-strand cross-links, and ssDNA gaps, as well as a pathway for DNA damage tolerance. HR differs from error-prone pathways for DNA damage repair/tolerance, such as nonhomologous end-joining (NHEJ) and translesion synthesis, in that it utilizes an intact, homologous duplex DNA as a donor to template the repair event. Moreover, many of the key intermediates in the HR pathway are reversible, allowing for exquisite regulation of the individual pathway steps. During the S, G2, and M phases of the cell cycle, HR competes with NHEJ for the repair of the two-ended DSBs 1 . In addition, HR is essential to DNA replication for the repair of replicationassociated DNA damage, including ssDNA gaps and one-sided DSBs, and as a mechanism of DNA lesion bypass 2 . A critical intermediate in the HR pathway is the displacement loop, or D-loop (Figure 1). Following end resection, the central recombinase in the reaction, Rad51, loads onto the newly resected ssDNA of the broken molecule, forming a helical filament 2 . Rad51 then carries out a homology search to identify a suitable homologous donor, typically the sister chromatid in somatic cells. The D-loop is formed when the Rad51-ssDNA filament invades a homologous duplex DNA, which leads to the Watson-Crick base pairing of the broken strand with the complementary strand of the donor, displacing the opposite donor strand. Extension of the 3' end of the broken strand by a DNA polymerase repl...

Members of the RecA family of strand exchange proteins carry out the central reaction in homologous recombination. These proteins are DNA-dependent ATPases, although their ATPase activity is not required for the key functions of homology search and strand exchange. We review the literature on the role of the intrinsic ATPase activity of strand exchange proteins. We also discuss the role of ATP-hydrolysis-dependent motor proteins that serve as strand exchange accessory factors, with an emphasis on the eukaryotic Rad54 family of double strand DNA-specific translocases. The energy from ATP allows recombination events to progress from the strand exchange stage to subsequent stages. ATP hydrolysis also functions to corrects DNA binding errors, including particularly detrimental binding to double strand DNA.