Frequency Of Homologous Recombination Research Articles

Chromosomal Translocations in the Parasite Leishmania by a MRE11/RAD50-Independent Microhomology-Mediated End Joining Mechanism.

The parasite Leishmania often relies on gene rearrangements to survive stressful environments. However, safeguarding a minimum level of genome integrity is important for cell survival. We hypothesized that maintenance of genomic integrity in Leishmania would imply a leading role of the MRE11 and RAD50 proteins considering their role in DNA repair, chromosomal organization and protection of chromosomes ends in other organisms. Attempts to generate RAD50 null mutants in a wild-type background failed and we provide evidence that this gene is essential. Remarkably, inactivation of RAD50 was possible in a MRE11 null mutant that we had previously generated, providing good evidence that RAD50 may be dispensable in the absence of MRE11. Inactivation of the MRE11 and RAD50 genes led to a decreased frequency of homologous recombination and analysis of the null mutants by whole genome sequencing revealed several chromosomal translocations. Sequencing of the junction between translocated chromosomes highlighted microhomology sequences at the level of breakpoint regions. Sequencing data also showed a decreased coverage at subtelomeric locations in many chromosomes in the MRE11-/-RAD50-/- parasites. This study demonstrates an MRE11-independent microhomology-mediated end-joining mechanism and a prominent role for MRE11 and RAD50 in the maintenance of genomic integrity. Moreover, we suggest the possible involvement of RAD50 in subtelomeric regions stability.

Establishing an efficient gene-targeting system in an itaconic-acid producing Aspergillus terreus strain.

To develop an efficient gene-targeting platform in an excellent itaconic acid producing strain Aspergillus terreus CICC40205. The frequency of homologous recombination was improved by deleting the ku80 gene. A nutritional transformation system based on the bidirectionally selectable marker, pyrG An , was established in the ku80-/pyrG-double mutant which is convenient for following marker rescue. The modified Cre/loxP recombination system was applied for the excision of the pyrG An marker by directly introducing Cre recombinase into the protoplasts. This gene-targeting system is an efficient platform for sequential and multiple genetic modifications in A. terreus and is conducive to study biosynthesis mechanisms and to improve the production ability of itaconic acid and other products.

The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation.

DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression.

Analysis of individual differences in radiosensitivity using genome editing.

Current standards for radiological protection of the public have been uniformly established. However, individual differences in radiosensitivity are suggested to exist in human populations, which could be caused by nucleotide variants of DNA repair genes. In order to verify if such genetic variants are responsible for individual differences in radiosensitivity, they could be introduced into cultured human cells for evaluation. This strategy would make it possible to analyse the effect of candidate nucleotide variants on individual radiosensitivity, independent of the diverse genetic background. However, efficient gene targeting in cultured human cells is difficult due to the low frequency of homologous recombination (HR) repair. The development of artificial nucleases has enabled efficient HR-mediated genome editing to be performed in cultured human cells. A novel genome editing strategy, 'transcription activator-like effector nuclease (TALEN)-mediated two-step single base pair editing', has been developed, and this was used to introduce a nucleotide variant associated with a chromosomal instability syndrome bi-allelically into cultured human cells to demonstrate that it is the causative mutation. It is proposed that this editing technique will be useful to investigate individual radiosensitivity.

A DNA2 Homolog Is Required for DNA Damage Repair, Cell Cycle Regulation, and Meristem Maintenance in Plants.

Plant meristem cells divide and differentiate in a spatially and temporally regulated manner, ultimately giving rise to organs. In this study, we isolated the Arabidopsis jing he sheng 1 (jhs1) mutant, which exhibited retarded growth, an abnormal pattern of meristem cell division and differentiation, and morphological defects such as fasciation, an irregular arrangement of siliques, and short roots. We identified JHS1 as a homolog of human and yeast DNA Replication Helicase/Nuclease2, which is known to be involved in DNA replication and damage repair. JHS1 is strongly expressed in the meristem of Arabidopsis. The jhs1 mutant was sensitive to DNA damage stress and had an increased DNA damage response, including increased expression of genes involved in DNA damage repair and cell cycle regulation, and a higher frequency of homologous recombination. In the meristem of the mutant plants, cell cycle progression was delayed at the G2 or late S phase and genes essential for meristem maintenance were misregulated. These results suggest that JHS1 plays an important role in DNA replication and damage repair, meristem maintenance, and development in plants.

Arabidopsis thaliana siRNA biogenesis mutants have the lower frequency of homologous recombination

ABSTRACTSmall interfering RNAs (siRNAs) are involved in the regulation of plant development and response to stress. We have previously shown that mutants impaired in Dicer-like 2 (DCL2), DCL3 and DCL4, RDR2, RDR6 and NPRD1 are partially impaired in their response to stress and dcl2 and dcl3 plants are also impaired in transgenerational response to stress, including changes in homologous recombination frequency (HRF). Here, we have analyzed genome stability of dcl2, dcl3, dcl4, dcl2 dcl3, dcl2 dcl3 dcl4 and rdr6 mutants by measuring the non-induced and the stress-induced recombination frequency. We found that all mutants had the lower spontaneous HRF. The analysis of strand breaks showed that all tested Arabidopsis mutants had a higher level of spontaneous strand breaks, suggesting that the lower HRF is not due to the unusually low level of breaks. Exposure to methyl methane sulfonate (MMS) resulted in an increase in the level of strand breaks in wild-type plants and a decrease in mutants. All mutants had the higher methylation of cytosines at CpG sites under non-induced conditions. Exposure to MMS resulted in a decrease in methylation level in wild-type plants and an increase in methylation in all dcl mutants. The expression of several DNA repair genes was altered in dcl4 plants under non-induced and induced conditions. Our data suggest that siRNA biogenesis may be essential for the maintenance of the genome stability and stress response in Arabidopsis.

Valproic acid causes radiosensitivity of breast cancer cells via disrupting the DNA repair pathway.

Valproic acid (VPA) is one of the representative compounds of histone deacetylase inhibitors (HDACis) and is used widely for the clinical treatment of epilepsy and other convulsive diseases. Current reports indicate that HDACis may also be an attractive radiosensitizer for some tumor cells; however, it is unknown whether the safe blood concentration of VPA (0.3-0.8 mM) used for the treatment of epilepsy can also induce radiosensitivity in breast cancer cells. In addition, the mechanism by which VPA may induce radiosensitivity in breast cancer cells is yet to be determined. Our results clearly indicated that VPA at a safe dose (0.5 mM) could significantly increase the radiosensitivity of MCF7 breast cancer cells and result in more accumulation of DNA double strand breaks in response to DNA damage. After VPA treatment, the frequencies of homologous recombination (HR) and non-homologous end joining (NHEJ) tested by recombination substrates, pDR-GFP and EJ5-GFP, were dramatically decreased in the cells without the change of the cell cycle profile. It was further found that VPA could inhibit the recruitment of key repair proteins to DNA break areas, such as Rad51, BRCA1, and Ku80. Thus, our results demonstrated that a safe dose of VPA causes radiosensitivity in breast cancer cells through disrupting the molecular mechanisms of both BRCA1-Rad51-mediated HR and Ku80-mediated NHEJ pathways.

Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

Analysis of the transgenerational iron deficiency stress memory in Arabidopsis thaliana plants.

We investigated the existence of the transgenerational memory of iron (Fe) deficiency stress, in Arabidopsis thaliana. Plants were grown under Fe deficiency/sufficiency, and so were their offspring. The frequency of somatic homologous recombination (SHR) events, of DNA strand breaks as well as the expression of the transcription elongation factor TFIIS-like gene increase when plants are grown under Fe deficiency. However, SHR frequency, DNA strand break events, and TFIIS-like gene expression do not increase further when plants are grown for more than one generation under the same stress, and furthermore, they decrease back to control values within two succeeding generations grown under control conditions, regardless of the Fe deficiency stress history of the mother plants. Seedlings produced from plants grown under Fe deficiency evolve more oxygen than control seedlings, when grown under Fe sufficiency: however, this trait is not associated with any change in the protein profile of the photosynthetic apparatus and is not transmitted to more than one generation. Lastly, plants grown for multiple generations under Fe deficiency produce seeds with greater longevity: however, this trait is not inherited in offspring generations unexposed to stress. These findings suggest the existence of multiple-step control of mechanisms to prevent a genuine and stable transgenerational transmission of Fe deficiency stress memory, with the tightest control on DNA integrity.

Exploitation of sulfonylurea resistance marker and non-homologous end joining mutants for functional analysis in Zymoseptoria tritici

The lack of techniques for rapid assembly of gene deletion vectors, paucity of selectable marker genes available for genetic manipulation and low frequency of homologous recombination are major constraints in construction of gene deletion mutants in Zymoseptoria tritici. To address these issues, we have constructed ternary vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici, which enable the single step assembly of multiple fragments via yeast recombinational cloning. The sulfonylurea resistance gene, which is a mutated allele of the Magnaporthe oryzae ILV2 gene, was established as a new dominant selectable marker for Z. tritici. To increase the frequency of homologous recombination, we have constructed Z. tritici strains deficient in the non-homologous end joining pathway of DNA double stranded break repair by inactivating the KU70 and KU80 genes. Targeted gene deletion frequency increased to more than 85% in both Z. tritici ku70 and ku80 null strains, compared to ⩽10% seen in the wild type parental strain IPO323. The in vitro growth and in planta pathogenicity of the Z. tritici ku70 and ku80 null strains were comparable to strain IPO323. Together these molecular tools add significantly to the platform available for genomic analysis through targeted gene deletion or promoter replacements and will facilitate large-scale functional characterization projects in Z. tritici.

DNA repair in plants studied by comet assay

Comet assay in plants.From the first description of the comet assay with isolated nuclei rather than whole cells it became evident that assay is well suited to be applied in plants (Koppen & Verschaeve, 1996). Disintegration of tissue by quick chopping with a razor blade, direct collection of released nuclei by patting and pipetting enables to process samples in time shorter than 2 minutes, the time prerequisite to study quick repair (Kozak et al, 2009).Plants are due to their sessile nature permanently exposed to environmental stresses (drought, salinity), ionizing (IR) and UV radiation and genotoxins, which directly or indirectly via generation of reactive oxidative species (ROS) damage their DNA. Radiomimetic Bleomycin functions as a catalyst to produce ROS leading to clusters of oxidized DNA lesions, single (SSB) and double (DSBs) strand breaks similarly as IR (Steighner & Povirk, 1990). Incurred SSBs and DSBs are easily distinguished and measured by comet assay when varying conditions of mainly used protocol with electrophoresis in 0.3 M NaOH, pH>13 solution (A/A assay). DSBs are detected under “neutral” conditions by N/N assay in regular electrophoretic buffer (Kozak et al, 2009; Olive & Banath, 2006), whilst SSBs are revealed by A/N assay, with alkali-unwinding step in 0.3 M NaOH prior electrophoresis (Angelis et al, 1999; Menke et al, 2001). Better resolution in DSBs and SSBs assays is observed when “neutral” conditions are set between pH 9-10, still well bellow DNA denaturing pH<11.6 (Bradley & Kohn, 1979).Repair of DSBs.DSBs are one of the most cytotoxic forms of DNA damage that must be repaired by recombination, predominantly via non-homologous joining of DNA ends (NHEJ) in higher eukaryotes. However, analysis of DSB repair kinetics of Arabidopsis NHEJ mutants atlig4 and atku80 with the N/N assay showed that repair of Bleomycin induced DSB is biphasic and rapid alternative repair pathways is active (Fig. 1). Surprisingly, kinetic measurements showed that rapid DSB repair was faster in the NHEJ mutant lines (t1/2 5.5 min.) than in wild-type Arabidopsis (t1/2 7.9 min.). Kozak et al. (Kozak et al, 2009) provided the first characterization of this alternate KU/LIG4-independent repair pathway that rapidly removes the majority of DSBs present in nuclear DNA and found its dependence on components of structural maintenance of chromosomes (SMC) complexes, namely SMC6b (MIM) of SMC5/6 complex, kleisin AtRAD21.1 and 3 of cohesin SMC1/3 complex (da Costa-Nunes et al, 2014) and structurally related SMCHD protein GMI1 (Bohmdorfer et al, 2011). Moreno-Romero et al. (Moreno-Romero et al, 2012) studied CK2 protein required for maintenance and control of genomic stability and used N/N assay to show that DSBs were more rapidly repaired in atck2 mutant than in control plants. Their results suggest that atck2 plants are more proficient in repairing DSBs and other lesions produced by IR or Bleomycin, despite their hypersensitivity to these agents.The moss Physcomitrella patens is unique besides the high frequency of homologous recombination for haploid state and filamentous growth during early stages of the vegetative growth. Sheared filaments enables to establish protonemal cultures with up to 50% dividing cells and makes Physcomitrella an excellent model plant to study DNA damage responses. Kamisugi et al. (Kamisugi et al, 2012) used N/N assay to study Physcomitrella mutants of pivotal DSB repair MRN complex (MRE11, RAD50, NBS1). Kinetics of DNA-DSB repair in wild-type and mutant plants revealed that Bleomycin-induced fragmentation of genomic DNA was quickly repaired at approximately equal rates (t1/2 3 min) in each genotype, although both, the ppmre11 and pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and Bleomycin-induced DNA damage. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; it is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development, the similar phenomenon as observed by Moreno-Romero et al. (Moreno-Romero et al, 2012) in atck2 plants. When MRN complex and CK2, both assumed to be associated with error-free homologous recombination are disabled, then NHEJ error-prone repair prevails at a cost of induced mutations in continuous DNA. And indeed this was proved by sequencing of mutated APT locus in which we identified deletions of various lengths as could be expected as an outcome of NHEJ repair mechanism. Combination of comet and mutation assays explained contradictory observation of rapid DSB repair in mutants with phenotype sensitive to induction of DSBs. Evidently plants evolved this attitude of rapid reconstitution of genome integrity from the early evolution stages (mosses were one of the first plants to colonize land) to higher eukaryote seed plants, where during every plant cycle embryos undergo severe DNA damage during seed maturation (generated by ROS released upon desiccation) and efficient recovery during seed germination.Repair of SSBs.

Generation of stable mutants and targeted gene deletion strains in Cryptococcus neoformans through electroporation.

Cryptococcus neoformans is the etiologic agent of cryptococcal meningitis that causes more than half a million deaths worldwide each year. This capsulated basidiomycetous yeast also serves as a model for micropathogenic studies. The ability to make stable mutants, either via ectopic integration or homologous recombination, has been accomplished using biolistic transformation. This technical advance has greatly facilitated the research on the basic biology and pathogenic mechanisms of this pathogen in the past two decades. However, biolistic transformation is costly, and its reproducibility varies widely. Here we found that stable ectopic integration or targeted gene deletion via homologous replacement could be accomplished through electroporative transformation. The stability of the transformants obtained through electroporation and the frequency of homologous replacement is highly dependent on the selective marker. A frequency of homologous recombination among the stable transformants obtained by electroporation is comparable to those obtained by biolistic transformation (∼10%) when dominant drug selection markers are used, which is much higher than what has been previously reported for electroporation when auxotrophic markers were used (0.001% to 0.1%). Furthermore, disruption of the KU80 gene or generation of gene deletion constructs using the split marker strategy, two approaches known to increase homologous replacement among transformants obtained through biolistic transformation, also increase the frequency of homologous replacement among transformants obtained through electroporation. Therefore, electroporation provides a low cost alternative for mutagenesis in Cryptococcus.

R-loop structure: the formation and the effects on genomic stability.

R-loop is a type of three-stranded nucleic acid structure that is made up of an RNA:DNA hybrid, formed due to failing separation of a nascent RNA molecule with transcripting template in transcription or by the re-annealing of RNA molecule with one of the two strands in a double stranded DNA molecule, along with the single stranded DNA, which is either the non-template strand in the transcription bubble or the RNA substituted DNA strand. Formation of R-loops can occur when transcription goes through a genomic DNA region having a tract of G bases in the non-template strand in the transcription bubble or through a type of triplet microsatellite DNA sequences that are known to be associated with certain human diseases. The negative supercoiling forces accumulated in the transcription bubble, and the misprocessing of RNA precursors, as well as the delayed utilizations and transportations of RNA molecules to cytoplasm promote R loop formation. Many studies show that cells can manage R loop formation with efficiency, and can also process the R-loops already formed in the cell, and by which, the bad effects of R-loops on DNA replication, gene mutation and homologous recombination can be regulated. In this review, we summarize the formation and the impacts of R-loops on DNA replication, mutation rates and the frequencies of homologous recombination, and also discusse the possible role of the R-loop induced DNA replication in mediating trinucleotide repeat expansions as seen with those frequently associated with human neuromuscular degenerative diseases.

Use of the meganuclease I-SceI of Saccharomyces cerevisiae to select for gene deletions in actinomycetes.

The search for new natural products is leading to the isolation of novel actinomycete species, many of which will ultimately require genetic analysis. Some of these isolates will likely exhibit low intrinsic frequencies of homologous recombination and fail to sporulate under laboratory conditions, exacerbating the construction of targeted gene deletions and replacements in genetically uncharacterised strains. To facilitate the genetic manipulation of such species, we have developed an efficient method to generate gene or gene cluster deletions in actinomycetes by homologous recombination that does not introduce any other changes to the targeted organism's genome. We have synthesised a codon optimised I-SceI gene for expression in actinomycetes that results in the production of the yeast I-SceI homing endonuclease which produces double strand breaks at a unique introduced 18 base pair recognition sequence. Only those genomes that undergo homologous recombination survive, providing a powerful selection for recombinants, approximately half of which possess the desired mutant genotype. To demonstrate the efficacy and efficiency of the system, we deleted part of the gene cluster for the red-pigmented undecylprodiginine complex of compounds in Streptomyces coelicolor M1141. We believe that the system we have developed will be broadly applicable across a wide range of actinomycetes.

Correlation between frequency of non-allelic homologous recombination and homology properties: evidence from homology-mediated CNV mutations in the human genome.

Non-allelic homologous recombination (NAHR) is one of the key mechanisms of DNA rearrangement. NAHR occurring between direct homologous repeats can generate genomic copy number variation (CNV) and make significant contributions to both genome evolution and human diseases such as cancer. Intriguingly, previous observations on the rare CNVs at certain genomic disorder loci suggested that NAHR frequency could be dependent on homology properties. However, such a correlation remains unclear at the other NAHR-mediated CNV loci, especially the common CNVs in human populations. Different from the rare CNVs associated with genomic disorders, it is challenging to identify de novo NAHR events at common CNV loci. Therefore, our previously proposed statistic M was employed in estimating relative mutation rate for the NAHR-mediated CNVs in human populations. By utilizing generalized regression neural network and principal component analysis in studying 4330 CNVs ascertained in 3 HapMap populations, we identified the CNVs mediated by NAHR between paired segmental duplications (SDs) and further revealed the correlations between SD properties and NAHR probability. SD length and inter-SD distance were shown to make major contributions to the occurrence of NAHR, whereas chromosomal position and sequence similarity of paired SDs are also involved in NAHR. An integrated effect of SD properties on NAHR frequency was revealed for the common CNVs in human populations. These observations can be well explained by ectopic synapsis in NAHR together with our proposed model of chromosomal compression/extension/looping (CCEL) for homology mis-pairing. Our findings showed the important roles of SDs in NAHR and human genomic evolution.

Rapid construction and screening of artificial microRNA systems in Chlamydomonas reinhardtii.

The unicellular green algae Chlamydomonas reinhardtii is a classic model for the study of flagella/cilia and photosynthesis, and it has recently been exploited for producing biopharmaceuticals and biofuel. Due to the low frequency of homologous recombination, reverse genetic manipulation in Chlamydomonas relies mainly on miRNA- and siRNA-based knockdown methods. However, the difficulty in constructing artificial miRNA vectors, laborious screening of knockdown transformants, and undesired epigenetic silencing of exogenous miRNA constructs limit their application. We have established a one-step procedure to construct an artificial miRNA precursor by annealing eight oligonucleotides of approximately 40 nucleotides. In the final construct, the Gaussia princeps luciferase gene (G-Luc) is positioned between the promoter and the artificial miRNA precursor so that knockdown strains may quickly be screened by visualizing luciferase luminescence using a photon-counting camera. Furthermore, the luciferase activity of transformants correlates with the knockdown level of two test target proteins: the chloroplast protein VIPP1 (vesicle inducing protein in plastids1) and the flagellar protein CDPK3 (calcium-dependent protein kinase3). Adding an intron from RBCS2 (ribulose bisphosphate carboxylase/oxygenase small subunit2) to the miRNA construct enhanced both the luciferase activity and the miRNA knockdown efficiency. A second miRNA vector incorporated the promoter of the nitrate reductase gene to allow inducible expression of the artificial miRNA. These vectors will facilitate application of the artificial miRNA and provide tools for studying the mechanism of epigenetics in Chlamydomonas, and may also be adapted for use in other model organisms.

A versatile and efficient markerless gene disruption system for Acidithiobacillus thiooxidans: application for characterizing a copper tolerance related multicopper oxidase gene.

The acidophilic bioleaching bacteria can usually survive in high concentrations of copper ions because of their special living environment. However, little is known about the copper homeostatic mechanisms of Acidithiobacillus thiooxidans, an important member of bioleaching bacteria. Here, a putative multicopper oxidase gene (cueO) was detected from the draft genome of A. thiooxidans ATCC 19377. The transcriptional level of cueO in response to 10 mM CuSO₄was upregulated 25.01 ± 2.59 folds. The response of P(cueO) to copper was also detected and might be stimulated by a putative CueR protein. Then, by using the counter-selectable marker lacZ and enhancing the expression of endonuclease I-SceI with tac promoter, a modified markerless gene disruption system was developed and the cueO gene disruption mutant (ΔcueO) of A. thiooxidans was successfully constructed with a markedly improved second homologous recombination frequency of 0.28 ± 0.048. The ΔcueO mutant was more sensitive to external copper and nearly completely lost the phenoloxidase activity; however, the activity could be restored after complementing the cueO gene. All results suggest the close relation of cueO gene to copper tolerance in A. thiooxidans. In addition, the developed efficient markerless gene knockout method can also be introduced into other Acidithiobacillus strains.

AtMMS21 regulates DNA damage response and homologous recombination repair in Arabidopsis

DNA damage is a significant problem in living organisms and DNA repair pathways have been evolved in different species to maintain genomic stability. Here we demonstrated the molecular function of AtMMS21, a component of SMC5/6 complex, in plant DNA damage response. Compared with wild type, the AtMMS21 mutant plants show hypersensitivity in the DNA damaging treatments by MMS, cisplatin and gamma radiation. However, mms21-1 is not sensitive to replication blocking agents hydroxyurea and aphidicolin. The expression of a DNA damage response gene PARP2 is upregulated in mms21-1 under normal condition, suggesting that this signaling pathway is constitutively activated in the mutant. Depletion of ATAXIA-TELANGIECTASIA MUTATED (ATM) in mms21-1 enhances its root growth defect phenotype, indicating that ATM and AtMMS21 may play additive roles in DNA damage pathway. The analysis of homologous recombination frequency showed that the number of recombination events is reduced in mms21-1 mutant. Conclusively, we provided evidence that AtMMS21 plays an important role in homologous recombination for DNA damage repair.

Heterogeneity in the frequency and characteristics of homologous recombination in pneumococcal evolution.

The bacterium Streptococcus pneumoniae (pneumococcus) is one of the most important human bacterial pathogens, and a leading cause of morbidity and mortality worldwide. The pneumococcus is also known for undergoing extensive homologous recombination via transformation with exogenous DNA. It has been shown that recombination has a major impact on the evolution of the pathogen, including acquisition of antibiotic resistance and serotype-switching. Nevertheless, the mechanism and the rates of recombination in an epidemiological context remain poorly understood. Here, we proposed several mathematical models to describe the rate and size of recombination in the evolutionary history of two very distinct pneumococcal lineages, PMEN1 and CC180. We found that, in both lineages, the process of homologous recombination was best described by a heterogeneous model of recombination with single, short, frequent replacements, which we call micro-recombinations, and rarer, multi-fragment, saltational replacements, which we call macro-recombinations. Macro-recombination was associated with major phenotypic changes, including serotype-switching events, and thus was a major driver of the diversification of the pathogen. We critically evaluate biological and epidemiological processes that could give rise to the micro-recombination and macro-recombination processes.

Mutagenic Organized Recombination Process by Homologous In Vivo Grouping (MORPHING) for Directed Enzyme Evolution

Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined segments of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein regions that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H2O2-sensitive versatile peroxidase by independent evolution of three distinct protein segments (Leu28-Gly57, Leu149-Ala174 and Ile199-Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence.