Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases.

In somatic mammalian cells, homologous recombination is a rare event. To study the effects of chromosomal breaks on frequency of homologous recombination, site-specific endonucleases were introduced into human cells by electroporation. Cell lines with a partial duplication within the HPRT (hypoxanthine phosphoribosyltransferase) gene were created through gene targeting. Homologous intrachromosomal recombination between the repeated regions of the gene can reconstruct a functioning, wild-type gene. Treatment of these cells with the restriction endonuclease Xba I, which has a recognition site within the repeated region of HPRT homology, increased the frequency or homologous recombination bv more than 10-fold. Recombination frequency was similarly increased by treatment with the rare-cutting yeast endonuclease PI-Sce I when a cleavage site was placed within the repeated region of HPRT. In contrast, four restriction enzymes that cut at positions either outside of the repeated regions or between them produced no change in recombination frequency. The results suggest that homologous recombination between intrachromosomal repeats can be specifically initiated by a double-strand break occurring within regions of homology, consistent with the predictions of a model.

BimD/SPO76 is at the interface of cell cycle progression, chromosome morphogenesis, and recombination

BIMD of Aspergillus nidulans belongs to a highly conserved protein family implicated, in filamentous fungi, in sister-chromatid cohesion and DNA repair. We show here that BIMD is chromosome associated at all stages, except from late prophase through anaphase, during mitosis and meiosis, and is involved in several aspects of both programs. First, bimD+ function must be executed during S through M. Second, in bimD6 germlings, mitotic nuclear divisions and overall cellular program occur more rapidly than in wild type. Thus, BIMD, an abundant chromosomal protein, is a negative regulator of normal cell cycle progression. Third, bimD6 reduces the level of mitotic interhomolog recombination but does not alter the ratio between crossover and noncrossover outcomes. Moreover, bimD6 is normal for intrachromosomal recombination. Therefore, BIMD is probably not involved in the enzymology of recombinational repair per se. Finally, during meiosis, staining of the Sordaria ortholog Spo76p delineates robust chromosomal axes, whereas BIMD stains all chromatin. SPO76 and bimD are functional homologs with respect to their roles in mitotic chromosome metabolism but not in meiosis. We propose that BIMD exerts its diverse influences on cell cycle progression as well as chromosome morphogenesis and recombination by modulating chromosome structure.

RecA protein stimulates homologous recombination in plants.

A number of RecA-like proteins have been found in eukaryotic organisms. We demonstrate that the prokaryotic recombination protein RecA itself is capable of interacting with genomic homologous DNA in somatic plant cells. Resistance to the DNA crosslinking agent mitomycin C requires homologous recombination as well as excision repair activity. Tobacco protoplasts expressing a nucleus-targeted RecA protein were at least three times as efficient as wild-type cells in repairing mitomycin C-induced damage. Moreover, homologous recombination at a defined locus carrying an endogenous nuclear marker gene was stimulated at least 10-fold in transgenic plant cells expressing nucleus-targeted RecA. The increase in resistance to mitomycin C and the stimulation of intrachromosomal recombination demonstrate that Escherichia coli RecA protein is functional in genomic homologous recombination in plants, especially when targeted to the plant nucleus.

Detection of Arabidopsis thaliana AtRAD1 cDNA variants and assessment of function by expression in a yeast rad1 mutant

The Saccharomyces cerevisiae RAD1 and human XPF genes encode a subunit of a nucleotide excision repair endonuclease that also is implicated in some forms of homologous recombination. An Arabidopsis thaliana gene (AtRAD1) encoding the orthologous plant protein has been identified recently. Here we report the isolation of three structurally distinct AtRAD1 cDNAs from A. thaliana leaf tissue RNA. One of the isolates (AtRAD1-1) corresponds to the cDNA previously shown to encode the full-length AtRad1 protein, whereas the other two (AtRAD1-2, AtRAD1-3) differ slightly in size due to variations at the 5′ end of exon 6 or the 3′ end of exon 7, respectively. The sequence differences argue that these cDNAs were probably templated by mRNAs generated via alternative splicing. Diagnostic polymerase chain reaction pointed to the presence of the AtRAD1-1 and AtRAD1-2 but not AtRAD1-3 transcripts in bud and root tissue, and to a fourth transcript (AtRAD1-4), having both alterations identified in AtRAD1-2 and AtRAD1-3, in root tissue. However, the low frequency of detection of AtRAD1-3 and AtRAD1-4 makes the significance of these tissue-specific patterns unclear. The predicted AtRad1-2, AtRad1-3 and AtRad1-4 proteins lack part of the region likely required for endonuclease complex formation. Expression of AtRAD1-2 and AtRAD1-3 in a yeast rad1 mutant did not complement the sensitivity to ultraviolet radiation or the recombination defect associated with the rad1 mutation. These results suggest that alternative splicing may modulate the levels of functional AtRad1 protein.

The role of XPF in recombinational repair

The DNA repair gene, XPF, is implicated in numerous processes relating to maintenance of genomic stability. The experiments presented herein were designed to investigate the role of XPF in homologous recombination processes. Specifically, the role of XPF in plasmid-chromosome and intrachromosomal recombination was evaluated. To interrogate the mechanistic role of XPF in plasmid-chromosome recombination, a homologous gene targeting system at the APRT locus in Chinese Hamster Ovary (CHO) cells was used. The targeting vector is linearized within 900 base pairs of heterology, which generates a substrate with long, nonhomologous 3′-OH ends that must be efficiently processed, presumably by the Xpf/Ercc1 heterodimer, prior to a productive recombination event. These experiments demonstrated a significant decrease in the targeted gene recombination frequency and a significant change to the recombinant product distributions in XPF- and ERCC1-deficient CHO cell lines, which suggest that the Xpf/Ercc1 heterodimer is essential for strand invasion recombination involving the processing of long, nonhomologous tails. In order to evaluate the role of XPF in intrachromosomal recombination, direct APRT repeat constructs at the chromosomal APRT locus in XPF-proficient and XPF-deficient CHO cells were used in spontaneous and DSB-induced recombination experiments. A defect in intrachromosomal recombination was only shown for UV41-derived XPF -deficient CHO cells, which have a severe interstrand crosslinking phenotype. The results of these studies demonstrate a requirement for XPF function in both plasmid-chromosome and intrachromosomal recombination, specifically in removal of long, single-stranded 3′-OH DNA ends. In addition, these studies identified a correlation between the interstrand cross-linking phenotype and the intrachromosomal recombination phenotype of each CHO cell line, but did not demonstrate a correlation between the interstrand cross-linking phenotype and the plasmid-chromosome recombination phenotype of these CHO cell lines. ^

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.

Electron diffusion and back reaction in dye-sensitized solar cells : the effect of nonlinear recombination kinetics

The electron collection efficiency in dye-sensitized solar cells (DSCs) is usually related to the electron diffusion length, L = (Dτ)^1/2, where D is the diffusion coefficient of mobile electrons and τ is their lifetime, which is determined by electron transfer to the redox electrolyte. Analysis of incident photon-to-current efficiency (IPCE) spectra for front and rear illumination consistently gives smaller values of L than those derived from small amplitude methods. We show that the IPCE analysis is incorrect if recombination is not first-order in free electron concentration, and we demonstrate that the intensity dependence of the apparent L derived by first-order analysis of IPCE measurements and the voltage dependence of L derived from perturbation experiments can be fitted using the same reaction order, γ ≈ 0.8. The new analysis presented in this letter resolves the controversy over why L values derived from small amplitude methods are larger than those obtained from IPCE data.

Kinetics of electron recombination of dye-senitized solar cells based on TiO2 nanorod arrays sensitized with different dyes

The performance and electron recombination kinetics of dye-sensitized solar cells based on TiO2 films consisting of one-dimensional nanorod arrays (NR-DSSCs) which are sensitized with dye N719, C218 and D205 respectively have been studied. It has been found that the best efficiency is obtained with the dye C218 based NR-DSSCs, benefiting from a 40% higher short-circuit photocurrent density. However, the open circuit photovoltage of the N719 based cell is 40 mV higher than that of the organic dye C218 and D205 based devices. Investigation of the electron recombination kinetics of the NR-DSSCs has revealed that the effective electron lifetime, τn, of the N719 based NR-DSSC is the lowest whereas the τn of the C218 based NR-DSSC is the highest among the three dyes. The higher Voc with the N719 based NR-DSSC is originated from the more negative energy level of the conduction band of the TiO2 film. In addition, in comparison to the DSSCs with conventional nanocrystalline particles based TiO2 films, the NR-DSSCs have shown over two orders of magnitude higher τn when employing N719 as the sensitizer. Nevertheless, the τn of the DSSCs with the C218 based nanorod arrays is only ten-fold higher than the that of the nanoparticles based devices. The remarkable characteristic of the dye C218 in suppressing the electron recombination of DSSCs is discussed.

Reduced electron recombination of dye-sensitized solar cells based on TiO 2 spheres consisting of ultrathin nanosheets with [001] facet exposed

An anatase TiO 2 material with hierarchically structured spheres consisting of ultrathin nanosheets with 100% of the [001] facet exposed was employed to fabricate dye-sensitized solar cells (DSC s). Investigation of the electron transport and back reaction of the DSCs by electrochemical impedance spectroscopy showed that the spheres had a threefold lower electron recombination rate compared to the conventional TiO 2 nanoparticles. In contrast, the effective electron diffusion coefficient, D n, was not sensitive to the variation of the TiO 2 morphology. The TiO 2 spheres showed the same Dn as that of the nanoparticles. The influence of TiCl 4 post-treatment on the conduction band of the TiO 2 spheres and on the kinetics of electron transport and back reactions was also investigated. It was found that the TiCl 4 post-treatment caused a downward shift of the TiO 2 conduction band edge by 30 meV. Meanwhile, a fourfold increase of the effective electron lifetime of the DSC was also observed after TiCl4 treatment. The synergistic effect of the variation of the TiO 2 conduction band and the electron recombination determined the open-circuit voltage of the DSC. © 2012 Wang et al.

Alkyl radical geometry controls geminate cage recombination in alkylcobalamins

Abstract: The radical pair that results from photolysis of adenosylcob(II1)alamin (AdoCbl"') undergoes primary geminate recombination with a first-order rate constant of 1 x lo9 s-l. In contrast, methylcob(II1)alamin (CH3Cbl"') and aristeromicylcob(II1)alamin (AriCblII', the carbocyclic analogue of AdoCbl"' in which the ribofuranose ring oxygen has been replaced with a methylene group) does not undergo primary geminate recombination. The ribofwanose group enables a high rate of geminate recombination in the [Ado' Cbl"'] radical pair. This may be due to a stereoelectronic (p-anomeric) effect that maintains a pyramidal geometry at the 5'-carbon of the 5'-deoxyadenosyl radical, or it may be due to hindered rotation about the C4t-C5, bond such that /?-elimination to the olefin is prevented. Recombination in the geminate singlet radical pair is in competition with diffusive escape to form a solvent-separated radical pair. Hyperfine coupling from Co" promotes intersystem crossing to the triplet radical pair (Chagovetz, A. M.; Grissom, C. B. J. Am. Chem. SOC. 1993, 115, 12152). Recombination of the [CH3' Cbl"] radical pair is not prevented by a lack of intersystem crossing, as neither unlabeled or I3C-labeled CH3Cbl"' undergoes geminate recombination. There is only a small difference in the rate of diffusive recombination in the solvent cage for AdoCbl"', AriCbl"', and CH3Cbl"' following photolysis: 2.01 x 10" s-l, 2.20 x lo4 s-l, and 1.16 x lo4 s-l. The rate of diffusive recombination is limited by productive collisions and not by radical geometry or intersystem crossing. The CF3' radical that results from photolysis of (trifluoromethyl)cob(III)alamin (CF3Cbl"') maintains its pyramidal geometry and undergoes faster diffusive recombination in the solvent cage at 51 x lo4 s-l. The C-Co bond dissociation enthalpy in AriCbl"' is 37 f 1.4 kcaymol. The profound difference in geminate recombination rates for AdoCbl"' and CH3Cbl"' is consistent with their different biological roles as enzymatic cofactors: AdoCbl"' is an initiator of radical chain chemistry in the active site, whereas CH3Cbl"' is a methyl group donor in an S~2-type process.

Recombination, decreased host specificity and increased mobility may have driven the emergence of maize streak virus as an agricultural pathogen

Maize streak virus (MSV; family Geminiviridae, genus Mastrevirus), the causal agent of maize streak disease, ranks amongst the most serious biological threats to food security in subSaharan Africa. Although five distinct MSV strains have been currently described, only one of these - MSV-A - causes severe disease in maize. Due primarily to their not being an obvious threat to agriculture, very little is known about the 'grass-adapted' MSV strains, MSV-B, -C, -D and -E. Since comparing the genetic diversities, geographical distributions and natural host ranges of MSV-A with the other MSV strains could provide valuable information on the epidemiology, evolution and emergence of MSV-A, we carried out a phylogeographical analysis of MSVs found in uncultivated indigenous African grasses. Amongst the 83 new MSV genomes presented here, we report the discovery of six new MSV strains (MSV-F to -K). The non-random recombination breakpoint distributions detectable with these and other available mastrevirus sequences partially mirror those seen in begomoviruses, implying that the forces shaping these breakpoint patterns have been largely conserved since the earliest geminivirus ancestors. We present evidence that the ancestor of all MSV-A variants was the recombinant progeny of ancestral MSV-B and MSV-G/-F variants. While it remains unknown whether recombination influenced the emergence of MSV-A in maize, our discovery that MSV-A variants may both move between and become established in different regions of Africa with greater ease, and infect more grass species than other MSV strains, goes some way towards explaining why MSV-A is such a successful maize pathogen. © 2008 SGM.

Evidence of ancient papillomavirus recombination

An open question amongst papillomavirus taxonomists is whether recombination has featured in the evolutionary history of these viruses. Since the onset of the global AIDS epidemic, the question is somewhat less academic, because immune-compromised human immunodeficiency virus patients are often co-infected with extraordinarily diverse mixtures of human papillomavirus (HPV) types. It is expected that these conditions may facilitate the emergence of HPV recombinants, some of which might have novel pathogenic properties. Here, a range of rigorous analyses is applied to full-genome sequences of papillomaviruses to provide convincing statistical and phylogenetic evidence that evolutionarily relevant papillomavirus recombination can occur. © 2006 SGM.

The evolutionary value of recombination is constrained by genome modularity

Genetic recombination is a fundamental evolutionary mechanism promoting biological adaptation. Using engineered recombinants of the small single-stranded DNA plant virus, Maize streak virus (MSV), we experimentally demonstrate that fragments of genetic material only function optimally if they reside within genomes similar to those in which they evolved. The degree of similarity necessary for optimal functionality is correlated with the complexity of intragenomic interaction networks within which genome fragments must function. There is a striking correlation between our experimental results and the types of MSV recombinants that are detectable in nature, indicating that obligatory maintenance of intragenome interaction networks strongly constrains the evolutionary value of recombination for this virus and probably for genomes in general.

Rapid host adaptation by extensive recombination

Experimental investigations into virus recombination can provide valuable insights into the biochemical mechanisms and the evolutionary value of this fundamental biological process. Here, we describe an experimental scheme for studying recombination that should be applicable to any recombinogenic viruses amenable to the production of synthetic infectious genomes. Our approach is based on differences in fitness that generally exist between synthetic chimaeric genomes and the wild-type viruses from which they are constructed. In mixed infections of defective reciprocal chimaeras, selection strongly favours recombinant progeny genomes that recover a portion of wild-type fitness. Characterizing these evolved progeny viruses can highlight both important genetic fitness determinants and the contribution that recombination makes to the evolution of their natural relatives. Moreover, these experiments supply precise information about the frequency and distribution of recombination breakpoints, which can shed light on the mechanistic processes underlying recombination. We demonstrate the value of this approach using the small single-stranded DNA geminivirus, maize streak virus (MSV). Our results show that adaptive recombination in this virus is extremely efficient and can yield complex progeny genomes comprising up to 18 recombination breakpoints. The patterns of recombination that we observe strongly imply that the mechanistic processes underlying rolling circle replication are the prime determinants of recombination breakpoint distributions found in MSV genomes sampled from nature. © 2009 SGM.