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  • br Development of the double strand


    Development of the double strand break repair model for meiotic recombination Meiotic recombination was first described by Frans Alfons Janssens using promotion in the salamander Batracoseps attenuatus in 1909 [21] and then further elaborated at the genetic level in Drosophila by Thomas H. Morgan [22]. In 1964, the British molecular biologist Robin Holliday, proposed a molecular model for meiotic recombination that featured symmetrical nicking and strand exchange between the recombining chromatids [23]. Lack of symmetry in the products of meiotic exchange led to the replacement of the Holliday model with the double strand break repair (DSBR) model 24., 25.. Subsequently, DSBs generated by the topoisomerase-like SPO11 was proposed to initiate meiotic recombination [26] (Fig. 2). The removal of SPO11 and its associated 5′ oligonucleotide is called ‘‘strand resection’’, a process that is carried out by the MRE11-RAD50-NBS1/XRS2 (MRN/X) complex to generate single-stranded 3′ overhangs (Fig. 2). The single-stranded DNA (ssDNA) is protected by replication protein A (RPA) [27]. RPA is then replaced by RecA-like proteins RAD51 [28] and meiosis-specific DMC1 [29] to form a nucleoprotein filament, which invades the homologous chromosome, displacing one strand and annealing to the other to create a D-loop and generate an intermediate joint molecule called a single end invasion (SEI). Using the invading 3′ end as a primer and its homologous stand as a template, the action of DNA synthesis extends the D-loop. Following DNA synthesis dependent D-loop expansion, sequences homologous to the 3′ single-stranded tail at the second side of the original DSB are exposed and can anneal in a process called second end capture. Additional DNA synthesis, and DNA ligation results in formation of a double Holliday Junction (dHJ) that is resolved to yield crossovers (COs) and, theoretically, non-crossovers (NCOs). In most organisms, only a minority of DSBs are processed via the DSBR pathway to form COs, while the vast majority ( ~95% in Arabidopsis) are processed as NCOs via an alternative pathway called the synthesis dependent strand annealing (SDSA) pathway. SDSA shares the same initial steps as DSBR, but instead of completing second end capture, the invading strand dissociates from the homologous chromatid, re-anneals to 3’ single-stranded tail at the opposite side of the original DSB, and undergoes gap-filling DNA synthesis to produce a NCO. The COs resulting from the DSBR pathway can further be divided into ZMM proteins (e.g. ZIP1, MSH4 and MER3)-dependent, interference sensitive (type I) pathway resolved from double Holliday Junction intermediate and a MUS81-dependent interference insensitive (type II) pathway [30] resolved from a single Holliday Junction intermediate (Fig. 2). Type I COs contribute ~85% of the total CO in budding yeast and Arabidopsis, while Type II COs contribute the remaining 15% 30., 31.. On the other hand, Type II COs are the only ones in S. pombe[32]. In the last several decades, molecular genetic studies have identified many genes required for different aspects of meiotic recombination using variety of model species 7., 33., 34., 35., 36.. However, few DNA synthesis components associated with meiotic recombination have been described. DNA synthesis during meiotic recombination may share factors with those required during DNA replication. As a result, lesions in these genes are potentially lethal to the embryo or other mitotic cells, making it difficult to study their roles in meiosis. It was observed that Arabidopsis male meiocytes express many DNA synthesis genes [37], suggesting a potential role for DNA synthesis factors in meiosis.
    The roles of DNA polymerases in meiotic recombination
    Formation of meiotic COs likely involves more DNA synthesis than NCO At several points, the DNA synthesis that occurs during recombination is primed from the 3′ end of one parental chromatid but uses a non-sister chromatid as a template. As a result, sequence information can be transferred between chromatids without the exchange of flanking markers (Fig. 2)—a process called gene conversion 54., 55.. Initial estimates using restriction endonuclease mapping of a single meiotic recombination hot spot in yeast suggested CO-associated conversion tracts (COCTs) and non-crossover-associated conversion tracts (NCOCTs) are ~1.4 and ~1.6kb respectively [56]. Higher resolution analysis using immunoprecipitation of 5-Bromo-2-deoxy Uridine (BrdU) labeled DNA suggested that COCTs and NCOCTs differ and are 1.9kb and 0.8kb, respectively [45]. Interestingly, these estimates agree with previous single-locus observation in humans [57] and mouse [58], and a subsequent genome-wide microarray study in budding yeast as well [59]. The development of high throughput sequencing enabled the analysis meiotic recombination in budding yeast within the four spores produced from individual meioses at single-base resolution [60]. Based on 46 thousand single nucleotide polymorphisms (SNPs), 91 COs and 21 NCOs were detected and maximum COCT lengths ranged from 164 to 10,637bp, while maximum NCOCT lengths ranged from 1,109 to 7,575bp. When the minimum size of the CO and NCO related gene conversion tract lengths were estimated, the related largest minimum sizes of CO and NCO are larger than 7kb and 6.5kb, respectively. These results infer that COCTs have various size, either less than 200bp, or rather extensive to several thousand bp.