Effects of identity minimization on Moloney murine leukemia virus template recognition and frequent tertiary template-directed insertions during nonhomologous recombination.
ABSTRACT: Homology requirements for Moloney murine leukemia virus recombination were addressed in this study by monitoring titer defects observed when acceptor/donor template identity lengths were systematically reduced. Recombination acceptors with at least 16 contiguous bases of donor template identity were recognized as efficiently as longer acceptors. In contrast, a sharp 1-log titer drop was observed for an acceptor of only 15 bases long, with an additional 1-log titer decline for an 8-base acceptor and further decreases for shorter acceptors. Eighty-three independent nonhomologous recombination products were sequenced to examine recombination template selection in the absence of significant sequence identity. These replication products contained a total of 152 nonhomologous crossover junctions. Forced copy choice models predict that forced nonhomologous recombination should result in DNA synthesis to the donor template's 5' end, followed by microidentity-guided acceptor template selection. However, only a single product displayed this structure. The majority of examined nonhomologous recombination products contained junction-associated sequence insertions. Most insertions resulted from the use of one or more tertiary templates, recognizable as discontiguous portions of viral or host RNA or minus-strand DNA. The donor/acceptor template microidentity evident at most crossovers reconfirmed the remarkable capability of the reverse transcription machinery to recognize short regions of sequence identity. These results demonstrate that recruitment of discontiguous host or viral sequences is a common way for retroviruses to resolve nonhomologous recombination junctions and provide experimental support for the role of splinting templates in the generation of retroviral insertions.
Project description:Class switch recombination (CSR) is a region-specific, transcriptionally regulated, nonhomologous recombinational process that is initiated by activation-induced cytidine deaminase (AID). The initial lesions in the switch (S) regions are subsequently processed and resolved, leading to recombination of the two targeted S regions. The mechanisms by which repair and ligation of the broken DNA ends occurs is still elusive. Recently, a small number of patients lacking DNA ligase IV, a critical component of the nonhomologous end joining (NHEJ) machinery, have been identified. We show that these patients display a considerably increased donor/acceptor homology at Smu-Salpha junctions compared with healthy controls. In contrast, Smu-Sgamma junctions show an increased frequency of insertions but no increase in junctional homology. These altered patterns of junctional resolution may be related to differences in the homology between the Smu and the downstream isotype S regions, and could reflect different modes of switch junction resolution when NHEJ is impaired. These findings link DNA ligase IV, and thus NHEJ, to CSR.
Project description:One model for retroviral transduction suggests that template switching between viral RNAs and polyadenylation readthrough sequences is responsible for the generation of acute transforming retroviruses. For this study, we examined reverse transcription products of human immunodeficiency virus (HIV)-based vectors designed to mimic postulated transduction intermediates. For maximization of the discontinuous mode of DNA synthesis proposed to generate transductants, sequences located between the vectors' two long terminal repeats (vector "body" sequences) and polyadenylation readthrough "tail" sequences were made highly homologous. Ten genetic markers were introduced to indicate which products had acquired tail sequences by a process we term transductive recombination. Marker segregation patterns for over 100 individual products were determined, and they revealed that more than half of the progeny proviruses were transductive recombinants. Although most crossovers occurred in regions of homology, about 5% were nonhomologous and some included insertions. Ratios of encapsidated readthrough and polyadenylated transcripts for vectors with wild-type and inactivated polyadenylation signals were compared, and transductive recombination frequencies were found to correlate with the readthrough transcript prevalence. In assays in which either vector body or tail could serve as a recombination donor, recombination between tail and body sequences was at least as frequent as body-body exchange. We propose that transductive recombination may contribute to natural HIV variation by providing a mechanism for the acquisition of nongenomic sequences.
Project description:Isotype switching involves a region-specific, nonhomologous recombinational deletion that has been suggested to occur by nonhomologous joining of broken DNA ends. Here, we find increased donor/acceptor homology at switch junctions from PMS2-deficient mice and propose that class switching can occur by microhomology-mediated end-joining. Interestingly, although isotype switching and somatic hypermutation show many parallels, we confirm that PMS2 deficiency has no major effect on the pattern of nucleotide substitutions generated during somatic hypermutation. This finding is in contrast to MSH2 deficiency. With MSH2, the altered pattern of switch recombination and hypermutation suggests parallels in the mechanics of the two processes, whereas the fact that PMS2 deficiency affects only switch recombination may reflect differences in the pathways of break resolution.
Project description:Perylene diimide (PDI) derivatives as a kind of promising non-fullerene-based acceptor (NFA) have got rapid development. However, most of the relevant developmental work has focused on synthesizing novel PDI-based structures, and few paid attentions to the selection of the polymer donor in PDI-based solar cells. Wide bandgap polymer (PBDB-T) and narrow bandgap polymer (PBDTTT-EFT) are known as the most efficient polymer donors in polymer solar cells (PSCs). While PBDB-T is in favor with non-fullerene acceptors achieving power conversion efficiency (PCE) more than 12%, PBDTTT-EFT is one of the best electron donors with fullerene acceptors with PCE up to 10%. Despite the different absorption profiles, the working principle of these benchmark polymer donors with a same electron acceptor, specially PDI-based acceptors, was rarely compared. To this end, we used PBDB-T and PBDTTT-EFT as the electron donors, and 1,1'-bis(2-methoxyethoxyl)-7,7'-(2,5-thienyl) bis-PDI (Bis-PDI-T-EG) as the electron acceptor to fabricate PSCs, and systematically compared their differences in device performance, carrier mobility, recombination mechanism, and film morphology.
Project description:The Rad51 (also known as RecA) family of recombinases executes the critical step in homologous recombination: the search for homologous DNA to serve as a template during the repair of DNA double-strand breaks (DSBs). Although budding yeast Rad51 has been extensively characterized in vitro, the stringency of its search and sensitivity to mismatched sequences in vivo remain poorly defined. Here, in Saccharomyces cerevisiae, we analysed Rad51-dependent break-induced replication in which the invading DSB end and its donor template share a 108-base-pair homology region and the donor carries different densities of single-base-pair mismatches. With every eighth base pair mismatched, repair was about 14% of that of completely homologous sequences. With every sixth base pair mismatched, repair was still more than 5%. Thus, completing break-induced replication in vivo overcomes the apparent requirement for at least 6-8 consecutive paired bases that has been inferred from in vitro studies. When recombination occurs without a protruding nonhomologous 3' tail, the mismatch repair protein Msh2 does not discourage homeologous recombination. However, when the DSB end contains a 3' protruding nonhomologous tail, Msh2 promotes the rejection of mismatched substrates. Mismatch correction of strand invasion heteroduplex DNA is strongly polar, favouring correction close to the DSB end. Nearly all mismatch correction depends on the proofreading activity of DNA polymerase-?, although the repair proteins Msh2, Mlh1 and Exo1 influence the extent of correction.
Project description:Nonfullerene solar cells have increased their efficiencies up to 13%, yet quantum efficiencies are still limited to 80%. Here we report efficient nonfullerene solar cells with quantum efficiencies approaching unity. This is achieved with overlapping absorption bands of donor and acceptor that increases the photon absorption strength in the range from about 570 to 700?nm, thus, almost all incident photons are absorbed in the active layer. The charges generated are found to dissociate with negligible geminate recombination losses resulting in a short-circuit current density of 20?mA?cm-2 along with open-circuit voltages >1?V, which is remarkable for a 1.6?eV bandgap system. Most importantly, the unique nano-morphology of the donor:acceptor blend results in a substantially improved stability under illumination. Understanding the efficient charge separation in nonfullerene acceptors can pave the way to robust and recombination-free organic solar cells.
Project description:The chemical structure of donors and acceptors limit the power conversion efficiencies achievable with active layers of binary donor-acceptor mixtures. Here, using quaternary blends, double cascading energy level alignment in bulk heterojunction organic photovoltaic active layers are realized, enabling efficient carrier splitting and transport. Numerous avenues to optimize light absorption, carrier transport, and charge-transfer state energy levels are opened by the chemical constitution of the components. Record-breaking PCEs of 18.07% are achieved where, by electronic structure and morphology optimization, simultaneous improvements of the open-circuit voltage, short-circuit current and fill factor occur. The donor and acceptor chemical structures afford control over electronic structure and charge-transfer state energy levels, enabling manipulation of hole-transfer rates, carrier transport, and non-radiative recombination losses.
Project description:The exchange of genetic information between donor and acceptor DNA molecules by homologous recombination (HR) depends on the cleavage of phosphodiester bonds. Although double-stranded and single-stranded DNA breaks (SSBs) have both been invoked as triggers of HR, until very recently the focus has been primarily on the former type of DNA lesions mainly due to the paucity of SSB-based recombination models. Here, to investigate the role of nicked DNA molecules as HR-initiating substrates in human somatic cells, we devised a homology-directed gene targeting system based on exogenous donor and chromosomal target DNA containing recognition sequences for the adeno-associated virus sequence- and strand-specific endonucleases Rep78 and Rep68. We found that HR is greatly fostered if a SSB is not only introduced in the chromosomal acceptor but also in the donor DNA template. Our data are consistent with HR models postulating the occurrence of SSBs or single-stranded gaps in both donor and acceptor molecules during the genetic exchange process. These findings can guide the development of improved HR-based genome editing strategies in which sequence- and strand-specific endonucleolytic cleavage of the chromosomal target site is combined with that of the targeting vector.
Project description:The reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retro-elements differ from retroviral RTs in being able to template switch from the 5' end of one template to the 3' end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3'-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. The rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3'-DNA overhang complementary to the 3' nucleotide of the acceptor RNA, suggesting a role for non-templated nucleotide addition of a complementary nucleotide to the 3’ end of cDNAs synthesized from natural templates. Longer 3'-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3' end of the acceptor template. Although dependent upon only a single base pair between the donor and acceptor, template switching discriminates against mismatches, which coupled with the high processivity of the enzyme, enables the synthesis of full-length DNA copies of acceptor nucleic acids beginning directly at their 3' end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTR-retroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq. Overall design: A group II intron RT was used to template switch and reverse transcribe synthetic RNA oligonucleotides and the resulting products were sequenced by Illumina Nextseq 500, and the junctions between acceptor and donor RNA molecules were analyzed.
Project description:CRISPR/Cas9, which generates DNA double-strand breaks (DSBs) at target loci, is a powerful tool for editing genomes when codelivered with a donor DNA template. However, DSBs, which are the most deleterious type of DNA damage, often result in unintended nucleotide insertions/deletions (indels) via mutagenic nonhomologous end joining. We developed a strategy for precise gene editing that does not generate DSBs. We show that a combination of single nicks in the target gene and donor plasmid (SNGD) using Cas9D10A nickase promotes efficient nucleotide substitution by gene editing. Nicking the target gene alone did not facilitate efficient gene editing. However, an additional nick in the donor plasmid backbone markedly improved the gene-editing efficiency. SNGD-mediated gene editing led to a markedly lower indel frequency than that by the DSB-mediated approach. We also show that SNGD promotes gene editing at endogenous loci in human cells. Mechanistically, SNGD-mediated gene editing requires long-sequence homology between the target gene and repair template, but does not require CtIP, RAD51, or RAD52. Thus, it is considered that noncanonical homology-directed repair regulates the SNGD-mediated gene editing. In summary, SNGD promotes precise and efficient gene editing and may be a promising strategy for the development of a novel gene therapy approach.