NHEJ deficiency develops homologous recombination in poplar meaningfully further than the overexpression of HDR factors

Efficient homology-directed DNA repair (HDR) is a vital difficulty confronting researchers to replace the target genome’s desired fragment. In plants, scientists have performed meticulous investigations on herbal, crops, and citrus trees using HDR effector proteins, CtIP and MRE11, to obtain double-stranded breaks (DSBs) more precisely. Although HDR efficiency in plants previously has been reported, no record has been declared about HDR efficiency in poplars.Here, we hypothesized that inhibition of nonhomologous recombination cofactors XRCC4, together with enhancing the HDR pathway activities, enables us to generate the HDR efficiency in poplar trees. In this study, the BleoR gene was used to integrate into the interested site and develop resistant poplars against Zeocin antibiotics. We designed plasmids, including different fusions of HDR proteins and, together with the XRCC4 target. Furthermore, real-time PCR, western blotting, RT-PCR, RT-qPCR, southern blotting, and DNA sequencing were applied to exhibit and evaluate HDR efficiency.While both applying HDR proteins and XRCC4 deficiency simultaneously could improve HDR efficiency, which showed about 50 times more than usual editing by CRISPR-Cas9, the only using HDR proteins without XRCC4 deficiency showed about 16 times more. We developed a new recombinant poplar genome to generate stable lines resistant to the Zeocin antibiotic.

Transpositional Recombination and Site-Specific Recombination May Be Initiated by Copy Choice during DNA Synthesis Rather Than Break/Join Mechanism

Types of DNA recombination include homologous recombination and nonhomologous recombination. Homologous DNA recombination is a general term that includes exchange of information between chromatids: (reciprocal) crossing-over, gene conversion, and post-meiotic segregation. Gene conversion is now thought to be a type of non-Mendelian segregation of heterozygous markers near the recombination initiation site. Thus, it includes both gene conversion and post-meiotic segregation previously described. DNA non-HR including transpositional recombination and site-specific recombination. Our understanding of the molecular mechanism by which DNA recombination occurs has significantly increased in the past decades. Currently The synthesis-dependent strand annealing model is now thought to give rise to most or all noncrossovers, with the double-strand-break repair model forming mainly crossovers. The Shapiro model proposed by Dr. J. Shapiro explains the molecular mechanism of transpositional recombination. Site-specific recombination results from another distinct model. We previously proposed a novel theory which can provide a more reasonable and simpler explanation accounting for DNA HR including the 3 classes of recombinogenic events described above. In the new supplementedly molecular model, DNA meiotic recombination can be initiated by a copy choice mechanism, that is, copying part of 1 single-stranded DNA template, followed by DNA polymerase switching to another single-stranded DNA template, and then resuming the following DNA synthesis along the new template. The current review suggests that transpositional recombination and site-specific recombination should be initiated by copy choice during DNA synthesis rather than break/join mechanism. The work indicates that review of DNA nonhomologous recombination are very necessary. The novel theory would challenge earlier models accounting for transpositional recombination and site-specific recombination and would be critical to the understanding of the mechanisms. We hope copy choice initiating DNA nonhomologous recombination will be one of the concepts that are explored. Proper and specific experiments are required to reconstruct the detailed mechanism described here.

CpG islands’ clustering uncovers early development genes in the human genome

We address the problem of the annotation of CpG islands (CGIs) clusters in the human genome. Upon analyzing gene content within CGIs clusters, piRNA, tRNA, and miRNA-encoding genes were found as well as CpG-rich homeobox genes reported previously. Chromosome-wide CGI density is positively correlated with replication timing, confirming that CGIs may serve as open chromatin markers. Early embryonic stage expressed KRAB-ZNF genes abundant at chromosome 19 were found to be interlinked with CGI clusters. We detected that a number of long CGIs and CGI clusters are, in fact, tandem copies with multiple annotated macrosatellites and paralogous genes. This finding implies that tandem expansion of CGIs may serve as a substrate for nonhomologous recombination events.

Tandem Duplicate Genes in Maize are Abundant and Date to Two Distinct Periods of Time

ABSTRACTTandem duplicate genes are proximally duplicated and as such occur in the same genomic neighborhood. Using the maize B73 and PH207 de novo genome assemblies, we identified thousands of tandem gene duplicates that account for ~10% of the genes. These tandem duplicates have a bimodal distribution of estimated ages corresponding to known periods of genomic instability. Tandem duplicates had a number of associated features that suggest origins in nonhomologous recombination based on smaller size distribution and higher rate of containing LTRs than non-tandem duplicates. Within relatively recent tandem duplicate genes, ~26% appear to be undergoing degeneration or divergence in function from the ancestral copy. Our results show that tandem duplicates are abundant in maize, arose in bursts throughout maize evolutionary history under multiple potential mechanisms, and may provide a substrate for novel phenotypic variation.

Genotype- and Cell-Specific Dynamics of Tandem Repeat Patterns in Aegilops speltoides Tausch (Poaceae, Triticeae)

In wild plant populations, chromosome rearrangements lead to the wide intraspecific polymorphisms in the abundance and patterns of highly repetitive DNA. However, despite the large amount of accumulated data, the impact of the complex repetitive DNA fraction on genome reorganization and functioning and the mechanisms balancing and maintaining the structural integrity of the genome are not fully understood. Homologous recombination is thought to play a key role in both genome reshuffling and stabilization, while the contribution of nonhomologous recombination seems to be undervalued. Here, tandem repeat patterns and dynamics during pollen mother cell development were addressed, with a focus on the meiotic recombination that determines chromosome/genome repatterning and stabilization under cross-pollination and artificial hybridization in wild goatgrass, Aegilops speltoides. Native plants from contrasting allopatric populations and artificially created intraspecific hybrids were investigated using a FISH approach. Cytogenetic analysis uncovered a wide spectrum of genotype- and cell-specific chromosomal rearrangements, suggesting intensive repatterning of both parental and hybrid genomes. The data obtained provide evidence that repetitive elements serve as overabundant and ubiquitous resources for maintaining chromosome architecture/genome integrity through homologous and nonhomologous recombination at the intraorganismal level, and genotype-specific repatterning underlies intrapopulation polymorphisms and intraspecific diversification in the wild.

Nonhomologous Recombination between Defective Poliovirus and Coxsackievirus Genomes Suggests a New Model of Genetic Plasticity for Picornaviruses

ABSTRACTMost of the circulating vaccine-derived polioviruses (cVDPVs) implicated in poliomyelitis outbreaks in Madagascar have been shown to be recombinants between the type 2 poliovirus (PV) strain of the oral polio vaccine (Sabin 2) and another species C human enterovirus (HEV-C), such as type 17 coxsackie A virus (CA17) in particular. We studied intertypic genetic exchanges between PV and non-PV HEV-C by developing a recombination model, making it possible to rescue defective type 2 PV RNA genomes with a short deletion at the 3′ end by the cotransfection of cells with defective or infectious CA17 RNAs. We isolated over 200 different PV/CA17 recombinants, using murine cells expressing the human PV receptor (PVR) and selecting viruses with PV capsids. We found some homologous (H) recombinants and, mostly, nonhomologous (NH) recombinants presenting duplications of parental sequences preferentially located in the regions encoding proteins 2A, 2B, and 3A. Short duplications appeared to be stable, whereas longer duplications were excised during passaging in cultured cells or after multiplication in PVR-transgenic mice, generating H recombinants with diverse sites of recombination. This suggests that NH recombination events may be a transient, intermediate step in the generation and selection of the fittest H recombinants. In addition to the classical copy-choice mechanism of recombination thought to generate mostly H recombinants, there may also be a modular mechanism of recombination, involving NH recombinant precursors, shaping the genomes of recombinant enteroviruses and other picornaviruses.IMPORTANCEThe multiplication of circulating vaccine-derived polioviruses (cVDPVs) in poorly immunized human populations can render these viruses pathogenic, causing poliomyelitis outbreaks. Most cVDPVs are intertypic recombinants between a poliovirus (PV) strain and another human enterovirus, such as type 17 coxsackie A viruses (CA17). For further studies of the genetic exchanges between PV and CA17, we have developed a model of recombination, making it possible to rescue defective PV RNA genomes with a short deletion by cotransfecting cells with the defective PV genome and CA17 genomic RNA. Numerous recombinants were found, including homologous PV/CA17 recombinants, but mostly nonhomologous recombinants presenting duplications of parental sequences preferentially located in particular regions. Long duplications were excised by passages in cultured cells or in mice, generating diverse homologous recombinants. Recombination leading to nonhomologous recombinants, which evolve into homologous recombinants, may therefore be seen as a model of genetic plasticity in enteroviruses and, possibly, in other RNA viruses.