Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries.

Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library's breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.

"Bridging the DNA divide": Understanding the interplay between replication- gaps and homologous recombination proteins RAD51 and BRCA1/2.

A key but often neglected component of genomic instability is the emergence of single-stranded DNA (ssDNA) gaps during DNA replication in the absence of functional homologous recombination (HR) proteins, such as RAD51 and BRCA1/2. Research in prokaryotes has shed light on the dual role of RAD51's bacterial ortholog, RecA, in HR and the protection of replication forks, emphasizing its essential role in preventing the formation of ssDNA gaps, which is vital for cellular viability. This phenomenon was corroborated in eukaryotic cells deficient in HR, where the formation of ssDNA gaps within newly synthesized DNA and their subsequent processing by the MRE11 nuclease were observed. Without functional HR proteins, cells employ alternative ssDNA gap-filling mechanisms to ensure survival, though this compensatory response can compromise genomic stability. A notable example is the involvement of the translesion synthesis (TLS) polymerase POLζ, along with the repair protein POLθ, in the suppression of replicative ssDNA gaps. Persistent ssDNA gaps may result in replication fork collapse, chromosomal anomalies, and cell death, which contribute to cancer progression and resistance to therapy. Elucidating the processes that avert ssDNA gaps and safeguard replication forks is critical for enhancing cancer treatment approaches by exploiting the vulnerabilities of cancer cells in these pathways.

The Cre/loxP-Based Recombinant HBV cccDNA System In Vitro and In Vivo.

Covalently closed circular DNA (cccDNA) exists as a stable episomal minichromosome in the nucleus of hepatocytes and is responsible for hepatitis B virus (HBV) persistence. We recently reported a technique involving recombinant cccDNA (rcccDNA) of HBV by site-specific DNA recombination. A floxed monomeric HBV genome was engineered into a precursor plasmid (prcccDNA) which was excised via Cre/loxP-mediated DNA recombination to form a 3.3-kb rcccDNA bearing a loxP-chimeric intron. The foreign sequence was efficiently removed during RNA splicing, rendering a functionally seamless insertion. We characterized rcccDNA formation, effective viral transcription, and replication induced by rcccDNA both in vitro and in vivo. Furthermore, we closely simulated chronic hepatitis by using a replication-defective recombinant adenoviral vector to deliver rcccDNA to the transgenic mice expressing Cre recombinase, which led to prominent HBV persistence. Here, we describe a detailed protocol about how to construct and evaluate Cre/loxP-based recombinant HBV cccDNA system both in vitro and in vivo.

Effects of demographic history on recombination hotspots in soybean.

Recombination is the primary mechanism underlying genetic improvement in populations and allows plant breeders to create new allelic combinations for agronomic improvement. Soybean [Glycine max (L.) Merr.] has gone through multiple genetic bottlenecks that have significantly affected its genetic diversity, linkage disequilibrium, and altered allele frequencies. To investigate the impact of genetic bottlenecks on recombination hotspots in soybeans, historical recombination was studied in three soybean populations. The populations were wild soybean [Glycine soja (Sieb. and Zucc.)], landraces, and North American elite soybean cultivars that have been genotyped with the SoySNP50K BeadChip. While each population after a genetic bottleneck had an increased average haplotype block size, they did not have a significant difference in the number of hotspots between each population. Instead, the increase in observed haplotype block size is likely due to an elimination of individuals that contained historical recombination at hotspots which decreased the observed rate of recombination for the hotspot after each genetic bottleneck. Conversely, heterochromatic DNA which has an increased haplotype block size compared to euchromatic DNA had a significantly different number of hotspots but not a significant difference in the average hotspot recombination rate. Previously identified genomic motifs associated with hotspots were also associated with hotspots found in the historical populations suggesting a common mechanism. This characterization of historical recombination hotspots in soybeans provides further insights into the effect genetic bottlenecks and selection have on recombination hotspots.

The recombination initiation functions DprA and RecFOR suppress microindel mutations in Acinetobacter baylyi ADP1.

Short-Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single-strands anneal with genomic DNA at microhomologies and get integrated during DNA replication, presumably acting as primers for Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR is tightly controlled by genome maintenance functions including RecA. It is thought that RecA scavenges DNA single-strands and renders them unable to anneal. To further elucidate the role of RecA in this process, we investigate the roles of the upstream functions DprA, RecFOR, and RecBCD, all of which load DNA single-strands with RecA. Here we show that all three functions suppress SPDIR mutations in the wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA, they are strongly increased in the absence of both DprA and RecA. This SPDIR-avoiding function of DprA is not related to its role in natural transformation. These results suggest a function for DprA in combination with RecA to avoid potentially harmful microindel mutations, and offer an explanation for the ubiquity of dprA in the genomes of naturally non-transformable bacteria.

Molecular insights into the prototypical single-stranded DNA-binding protein from E. coli.

The SSB protein of Escherichia coli functions to bind single-stranded DNA wherever it occurs during DNA metabolism. Depending upon conditions, SSB occurs in several different binding modes. In the course of its function, SSB diffuses on ssDNA and transfers rapidly between different segments of ssDNA. SSB interacts with many other proteins involved in DNA metabolism, with 22 such SSB-interacting proteins, or SIPs, defined to date. These interactions chiefly involve the disordered and conserved C-terminal residues of SSB. When not bound to ssDNA, SSB can aggregate to form a phase-separated biomolecular condensate. Current understanding of the properties of SSB and the functional significance of its many intermolecular interactions are summarized in this review.

Deficiency of both classical and alternative end-joining pathways leads to a synergistic defect in double-strand break repair but not to an increase in homology-dependent gene targeting in Arabidopsis.

In eukaryotes, double-strand breaks (DSBs) are either repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). In somatic plant cells, HR is very inefficient. Therefore, the vast majority of DSBs are repaired by two different pathways of NHEJ. The classical (cNHEJ) pathway depends on the heterodimer KU70/KU80, while polymerase theta (POLQ) is central to the alternative (aNHEJ) pathway. Surprisingly, Arabidopsis plants are viable, even when both pathways are impaired. However, they exhibit severe growth retardation and reduced fertility. Analysis of mitotic anaphases indicates that the double mutant is characterized by a dramatic increase in chromosome fragmentation due to defective DSB repair. In contrast to the single mutants, the double mutant was found to be highly sensitive to the DSB-inducing genotoxin bleomycin. Thus, both pathways can complement for each other efficiently in DSB repair. We speculated that in the absence of both NHEJ pathways, HR might be enhanced. This would be especially attractive for gene targeting (GT) in which predefined changes are introduced using a homologous template. Unexpectedly, the polq single mutant as well as the double mutant showed significantly lower GT frequencies in comparison to wildtype plants. Accordingly, we were able to show that elimination of both NHEJ pathways does not pose an attractive approach for Agrobacterium-mediated GT. However, our results clearly indicate that a loss of cNHEJ leads to an increase in GT frequency, which is especially drastic and attractive for practical applications, in which the in planta GT strategy is used.

Insertion sequence excision is enhanced by a protein that catalyzes branch migration and promotes microhomology-mediated end joining.

Insertion sequence (IS)-excision enhancer (IEE) promotes the excision of ISs in the genome of enterohemorrhagic Escherichia coli O157. Because IEE-dependent IS excision occurs in the presence of transposase, the process of IS transposition may be involved in IS excision; however, little is understood about the molecular mechanisms of IS excision. Our in vitro analysis revealed that IEE exhibits DNA-dependent ATPase activity, which is activated by branched DNA. IEE also catalyzes the branch migration of fork-structured DNA. These results suggest that IEE remodels branched structures of the IS transposition intermediate. Sequence analysis of recombination sites in IS-excision products suggested that microhomologous sequences near the ends of the IS are involved in IS excision. IEE promoted microhomology-mediated end joining (MMEJ), in which base pairing between 6-nucleotides complementary ends of two 3'-protruding DNAs and subsequent elongation of the paired DNA strand occurred. IS-excision frequencies were significantly decreased in cells producing IEE mutants that had lost either branch migration or MMEJ activity, which suggests that these activities of IEE are required for IS excision. Based on our results, we propose a model for IS excision triggered by IEE and transposase.

Telomere maintenance in African trypanosomes.

Telomere maintenance is essential for genome integrity and chromosome stability in eukaryotic cells harboring linear chromosomes, as telomere forms a specialized structure to mask the natural chromosome ends from DNA damage repair machineries and to prevent nucleolytic degradation of the telomeric DNA. In Trypanosoma brucei and several other microbial pathogens, virulence genes involved in antigenic variation, a key pathogenesis mechanism essential for host immune evasion and long-term infections, are located at subtelomeres, and expression and switching of these major surface antigens are regulated by telomere proteins and the telomere structure. Therefore, understanding telomere maintenance mechanisms and how these pathogens achieve a balance between stability and plasticity at telomere/subtelomere will help develop better means to eradicate human diseases caused by these pathogens. Telomere replication faces several challenges, and the "end replication problem" is a key obstacle that can cause progressive telomere shortening in proliferating cells. To overcome this challenge, most eukaryotes use telomerase to extend the G-rich telomere strand. In addition, a number of telomere proteins use sophisticated mechanisms to coordinate the telomerase-mediated de novo telomere G-strand synthesis and the telomere C-strand fill-in, which has been extensively studied in mammalian cells. However, we recently discovered that trypanosomes lack many telomere proteins identified in its mammalian host that are critical for telomere end processing. Rather, T. brucei uses a unique DNA polymerase, PolIE that belongs to the DNA polymerase A family (E. coli DNA PolI family), to coordinate the telomere G- and C-strand syntheses. In this review, I will first briefly summarize current understanding of telomere end processing in mammals. Subsequently, I will describe PolIE-mediated coordination of telomere G- and C-strand synthesis in T. brucei and implication of this recent discovery.

How RNA impacts DNA repair.

The central dogma of molecular biology posits that genetic information flows unidirectionally, from DNA, to RNA, and finally to protein. However, this directionality is broken in some cases, such as reverse transcription where RNA is converted to DNA by retroviruses and certain transposable elements. Our genomes have evolved and adapted to the presence of reverse transcription. Similarly, our genome is continuously maintained by several repair pathways to reverse damage due to various endogenous and exogenous sources. More recently, evidence has revealed that RNA, while in certain contexts may be detrimental for genome stability, is involved in promoting certain types of DNA repair. Depending on the pathway in question, the size of these DNA repair-associated RNAs range from one or a few ribonucleotides to long fragments of RNA. Moreover, RNA is highly modified, and RNA modifications have been revealed to be functionally associated with specific DNA repair pathways. In this review, we highlight aspects of this unexpected layer of genomic maintenance, demonstrating how RNA may influence DNA integrity.

Holliday junction branch migration driven by AAA+ ATPase motors.

Holliday junctions are key intermediate DNA structures during genetic recombination. One of the first Holliday junction-processing protein complexes to be discovered was the well conserved RuvAB branch migration complex present in bacteria that mediates an ATP-dependent movement of the Holliday junction (branch migration). Although the RuvAB complex served as a paradigm for the processing of the Holliday junction, due to technical limitations the detailed structure and underlying mechanism of the RuvAB branch migration complex has until now remained unclear. Recently, structures of a reconstituted RuvAB complex actively-processing a Holliday junction were resolved using time-resolved cryo-electron microscopy. These structures showed distinct conformational states at different stages of the migration process. These structures made it possible to propose an integrated model for RuvAB Holliday junction branch migration. Furthermore, they revealed unexpected insights into the highly coordinated and regulated mechanisms of the nucleotide cycle powering substrate translocation in the hexameric AAA+ RuvB ATPase. Here, we review these latest advances and describe areas for future research.

[Development of a tau-V337M mouse model using CRISPR/Cas9 system and enhanced ssODN-mediated recombination].

The generation of a tau-V337M point mutation mouse model using gene editing technology can provide an animal model with fast disease progression and more severe symptoms, which facilitate the study of pathogenesis and treatment of Alzheimer's disease (AD). In this study, single guide RNAs (sgRNA) and single-stranded oligonucleotides (ssODN) were designed and synthesized in vitro. The mixture of sgRNA, Cas9 protein and ssODN was microinjected into the zygotes of C57BL/6J mice. After DNA cutting and recombination, the site homologous to human 337 valine (GTG) in exon 11 was mutated into methionine (ATG). In order to improve the efficiency of recombination, a Rad51 protein was added. The female mice mated with the nonvasectomy male mice were used as the surrogates. Subsequently, the 2-cell stage gene edited embryos were transferred into the unilateral oviduct, and the F0 tau-V337M mutation mice were obtained. Higher mutation efficiency could be obtained by adding Rad51 protein. The F0 tau-V337M point mutation mice can pass the mutation on to the F1 generation mice. In conclusion, this study successfully established the first tau-V337M mutation mouse by using Cas9, ssODN and Rad51. These results provide a new method for developing AD mice model which can be used in further research on the pathogenesis and treatment of AD.

Establishment and characterization of a novel reverse genetic system of BK polyomavirus.

BK polyomavirus (BKV) is a small non-enveloped DNA virus. BKV infection or reactivation may cause BKV-associated nephropathy and hemorrhagic cystitis in immunosuppressed transplant recipients. No effective antivirals or prevention strategies are available against BKV infections. The current BKV reverse system employs the transfection of purified full-length linear viral genomes released by enzyme digestion from BKV genomic plasmids. The method is laborious and often results in variable DNA yield and quality, which can affect the efficiency of transfection and subsequent formation of circular viral genomes in cells. In this study, we report the generation of circular viral genomes by Cre-mediated DNA recombination in cells directly transfected with BKV precursor genomic plasmids. The novel system supported efficient viral expression and replication, and produced a higher level of infectious virions compared with the transfection with linear BKV genomes. Furthermore, we successfully constructed recombinant BKV capable of reporter gene expression. In conclusion, the novel BKV reverse genetic system allows for simpler manipulation of BKV genome with better virus yield, providing a tool for the study of BKV life cycle and antiviral screening.

Swc4 protects nucleosome-free rDNA, tDNA and telomere loci to inhibit genome instability.

In the baker's yeast Saccharomyces cerevisiae, NuA4 and SWR1-C, two multisubunit complexes, are involved in histone acetylation and chromatin remodeling, respectively. Eaf1 is the assembly platform subunit of NuA4, Swr1 is the assembly platform and catalytic subunit of SWR1-C, while Swc4, Yaf9, Arp4 and Act1 form a functional module, and is present in both NuA4 and SWR1 complexes. ACT1 and ARP4 are essential for cell survival. Deletion of SWC4, but not YAF9, EAF1 or SWR1 results in a severe growth defect, but the underlying mechanism remains largely unknown. Here, we show that swc4Δ, but not yaf9Δ, eaf1Δ, or swr1Δ cells display defects in DNA ploidy and chromosome segregation, suggesting that the defects observed in swc4Δ cells are independent of NuA4 or SWR1-C integrity. Swc4 is enriched in the nucleosome-free regions (NFRs) of the genome, including characteristic regions of RDN5s, tDNAs and telomeres, independently of Yaf9, Eaf1 or Swr1. In particular, rDNA, tDNA and telomere loci are more unstable and prone to recombination in the swc4Δ cells than in wild-type cells. Taken together, we conclude that the chromatin associated Swc4 protects nucleosome-free chromatin of rDNA, tDNA and telomere loci to ensure genome integrity.

A simple, dual direct expression plasmid system in prokaryotic and mammalian cells.

We introduce a simple, dual direct cloning plasmid system (pgMAX-II) for gene expression analysis in both prokaryotic (Escherichia coli) and mammalian cells. This system, which uses a prokaryotic expression unit adapted from the pgMAX system and a mammalian promoter, is effective for subcloning using the DNA topoisomerase II toxin CcdB. Given that molecular biological cloning systems broadly rely on E. coli for rapid growth, the proposed concept may have wide applicability beyond mammalian cells.

Generation and Repair of Postreplication Gaps in Escherichia coli.

When replication forks encounter template lesions, one result is lesion skipping, where the stalled DNA polymerase transiently stalls, disengages, and then reinitiates downstream to leave the lesion behind in a postreplication gap. Despite considerable attention in the 6 decades since postreplication gaps were discovered, the mechanisms by which postreplication gaps are generated and repaired remain highly enigmatic. This review focuses on postreplication gap generation and repair in the bacterium Escherichia coli. New information to address the frequency and mechanism of gap generation and new mechanisms for their resolution are described. There are a few instances where the formation of postreplication gaps appears to be programmed into particular genomic locations, where they are triggered by novel genomic elements.

Polymorphism of Saccharomyces cerevisiae Strains in DNA Metabolism Genes.

Baker's yeast, S. cerevisiae, is an excellent model organism exploited for molecular genetic studies of the mechanisms of genome stability in eukaryotes. Genetic peculiarities of commonly used yeast strains impact the processes of DNA replication, repair, and recombination (RRR). We compared the genomic DNA sequence variation of the five strains that are intensively used for RRR studies. We used yeast next-generation sequencing data to detect the extent and significance of variation in 183 RRR genes. We present a detailed analysis of the differences that were found even in closely related strains. Polymorphisms of common yeast strains should be considered when interpreting the outcomes of genome stability studies, especially in cases of discrepancies between laboratories describing the same phenomena.

The regulation of meiotic crossover distribution: a coarse solution to a century-old mystery?

Meiotic crossovers, which are exchanges of genetic material between homologous chromosomes, are more evenly and distantly spaced along chromosomes than expected by chance. This is because the occurrence of one crossover reduces the likelihood of nearby crossover events - a conserved and intriguing phenomenon called crossover interference. Although crossover interference was first described over a century ago, the mechanism allowing coordination of the fate of potential crossover sites half a chromosome away remains elusive. In this review, we discuss the recently published evidence supporting a new model for crossover patterning, coined the coarsening model, and point out the missing pieces that are still needed to complete this fascinating puzzle.

Rapid method for plasmid DNA recombination (Murakami-system).

DNA recombination is a useful technology for cloning and subsequent functional analysis, while standard techniques for plasmid DNA recombination have remained unchanged. In the present study, we introduced rapid method for plasmid DNA recombination, which we named "Murakami-system", to complete the experiments in under 33 h. For this purpose, we selected the following: PCR amplification with 25 cycles and E. coli strain with rapid growth (incubation time of 6-8 h). In addition, we selected rapid plasmid DNA purification (mini-prep; ∼10 min) and rapid restriction enzyme incubation (20 min). This recombination system enabled rapid plasmid DNA recombination within 24-33 h, which could be useful in various fields. We also established a 1-day method for competent cell preparation. Our rapid recombination system allowed several sessions of plasmid DNA recombination to be performed every week, which improves the functional analysis of various genes.•"Rapid method for plasmid DNA recombination (Murakami-system).•E. coli strain with rapid growth (incubation time of 6-8 h).•Combination of rapid protocols (PCR, electrophoresis, DNA purification, ligation, and mini-prep) enabled plasmid DNA recombination within 24-33 h.