Choreography of recombination proteins during the DNA damage response

Genome integrity is frequently challenged by DNA lesions from both endogenous and exogenous sources. A single DNA double-strand break (DSB) is lethal if unrepaired and may lead to loss of heterozygosity, mutations, deletions, genomic rearrangements and chromosome loss if repaired improperly. Such genetic alterations are the main causes of cancer and other genetic diseases. Consequently, DNA double-strand break repair (DSBR) is an important process in all living organisms. DSBR is also the driving mechanism in most strategies of gene targeting, which has applications in both genetic and clinical research. Here we review the cell biological response to DSBs in mitotically growing cells with an emphasis on homologous recombination pathways in yeast Saccharomyces cerevisiae and in mammalian cells.     

Illumina mate-paired DNA sequencing-library preparation using Cre-Lox recombination

Standard Illumina mate-paired libraries are constructed from 3- to 5-kb DNA fragments by a blunt-end circularization. Sequencing reads that pass through the junction of the two joined ends of a 3-5-kb DNA fragment are not easy to identify and pose problems during mapping and de novo assembly. Longer read lengths increase the possibility that a read will cross the junction. To solve this problem, we developed a mate-paired protocol for use with Illumina sequencing technology that uses Cre-Lox recombination instead of blunt end circularization. In this method, a LoxP sequence is incorporated at the junction site. This sequence allows screening reads for junctions without using a reference genome. Junction reads can be trimmed or split at the junction. Moreover, the location of the LoxP sequence in the reads distinguishes mate-paired reads from spurious paired-end reads. We tested this new method by preparing and sequencing a mate-paired library with an insert size of 3 kb from Saccharomyces cerevisiae. We present an analysis of the library quality statistics and a new bio-informatics tool called DeLoxer that can be used to analyze an IlluminaCre-Lox mate-paired data set. We also demonstrate how the resulting data significantly improves a de novo assembly of the S. cerevisiae genome.     

Molecular recognition of DNA structure by proteins that mediate genetic recombination

The latter half of genetic recombination is mediated by proteins that recognise the structure of the four-way DNA junction, and manipulate this structure. In solution the four-way junction adopts a stacked X-structure in the presence of metal ions. The folding is brought about by the pairwise coaxial stacking of helices in a right-handed antiparallel X-shaped structure. The four-way junction is cleaved by structure-selective resolving enzymes that have been isolated from a wide variety of sources, from eubacteria and their phages through to mammals. In addition, another class of proteins accelerate the branch migration of the junction. These proteins all appear to be divisible into a component that recognises structure and another that carries out a reaction on the junction. Thus the ability of structure-selective binding to the four-way DNA junction is a key feature of enzymes important in genetic recombination.     

Automated phylogenetic detection of recombination using a genetic algorithm

The evolution of homologous sequences affected by recombination or gene conversion cannot be adequately explained by a single phylogenetic tree. Many tree-based methods for sequence analysis, for example, those used for detecting sites evolving nonneutrally, have been shown to fail if such phylogenetic incongruity is ignored. However, it may be possible to propose several phylogenies that can correctly model the evolution of nonrecombinant fragments. We propose a model-based framework that uses a genetic algorithm to search a multiple-sequence alignment for putative recombination break points, quantifies the level of support for their locations, and identifies sequences or clades involved in putative recombination events. The software implementation can be run quickly and efficiently in a distributed computing environment, and various components of the methods can be chosen for computational expediency or statistical rigor. We evaluate the performance of the new method on simulated alignments and on an array of published benchmark data sets. Finally, we demonstrate that prescreening alignments with our method allows one to analyze recombinant sequences for positive selection.     

Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae

Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4 Delta strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase alpha (Pol alpha), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G(2)-arrested cells. Whereas PCNA was still essential for MAT switching, neither Pol alpha nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Pol alpha-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.     

VisRD--visual recombination detection

SUMMARY: VisRD, a program for visual recombination detection in a sequence alignment is presented. VisRD is written in Java and is designed to complement the multi-purpose phylogenetic software package SplitsTree4. AVAILABILITY: The software is freely available from http://www.lcb.uu.se/~vmoulton/software/visrd/     

The interaction of recombination proteins with supercoiled DNA: defining the role of supercoiling in lambda integrative recombination

Lambda integrative recombination depends on supercoiling of the phage attachment site, attP. Using dimethylsulfate protection and indirect end-labeling, the interaction of the recombination proteins Int and IHF with supercoiled and linear attP has been studied. Supercoiling enhances the binding of Int to attP, but not if a truncated attP site is employed or if IHF is omitted. We reason that the altered affinity reflects the formation of a higher-order nucleoprotein structure, an "attP intasome," that involves Int and IHF assembly of both arms of attP into a wrapped configuration. The good correlation between the degree and sign of supercoiling needed to promote recombination and that needed for the "attP intasome" indicates that the primary role of supercoiling is to drive the formation of the wrapped structure.     

Sensitive detection of proteins using assembled cascade fluorescent DNA nanotags based on rolling circle amplification

A novel cascade fluorescence signal amplification strategy based on the rolling circle amplification (RCA)-aided assembly of fluorescent DNA nanotags as fluorescent labels and multiplex binding of the biotin-streptavidin system was proposed for detection of protein target at ultralow concentration. In the strategy, fluorescent DNA nanotags are prepared relying on intercalating dye arrays assembled on nanostructured DNA templates by intercalation between base pairs. The RCA product containing tandem-repeat sequences could serve as an excellent template for periodic assembly of fluorescent DNA nanotags, which were presented per protein recognition event to numerous fluorescent DNA nanotags for assay readout. Both the RCA and the multiplex binding system showed remarkable amplification efficiency, very little nonspecific adsorption, and low background signal. Using human IgG as a model protein, the designed strategy was successfully demonstrated for the ultrasensitive detection of protein target. The results revealed that the strategy exhibited a dynamic response to human IgG over a three-decade concentration range from 1.0 pM to 1.0 fM with a limit of detection as low as 0.9 fM. By comparison with the assay of multiple labeling antibodies with the dye/DNA conjugate, the limit of detection was improved by 4 orders. The designed signal amplification strategy would hold great promise as a powerful tool to be applied for the ultrasensitive detection of target protein in immunoassay.     

Label-free detection of proteins using SERS-based immuno-nanosensors

This paper describes the development and optimization of a novel class of SERS-based immuno-nanosensors for the label-free detection of specific proteins in complex environments (e.g., cell culture matrices and intracellular environments). These SERS-based nanosphere sensors are fabricated by depositing multiple layers of silver on silica nanospheres, followed by binding of the antibody of interest to the silver surface via a short rigid crosslinker. In these studies, several different crosslinkers were characterized and evaluated for optimal nanosensor activity. The crosslinkers evaluated contained either thiol or isothiocyanate functionalities, which bind to the silver surface on one end, while the other end of the crosslinker contained either a carboxylic or primary amine group, which reacts readily with the antibodies. These SERS-based nanosensors were also optimized for underlying silica sphere diameters, silica sphere coating conditions during silver deposition, number of silver layers applied, and silver surface coverage with crosslinkers. Upon optimization, the nanosensors were evaluated by monitoring their response to various antigens (e.g., human insulin or interleukin II) in complex environments.     

Label-free DNA sequencing using Millikan detection

A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucleotides attached to the bead. The velocity of beads tethered via a polymer to a microfluidic channel and subjected to an oscillating electric field was measured using dark-field microscopy and used to determine how many nucleotides were incorporated during each sequencing-by-synthesis cycle. Increases in bead velocity of approximately 1% were reliably detected during DNA polymerization, allowing for sequencing of short DNA templates. The method could lead to a low-cost, high-throughput sequencing platform that could enable routine sequencing in medical applications.     

DNA detection using commercial mobile phones

This study investigates the feasibility of using mobile phones cameras for DNA detection. DNA amplification uses the convective polymerase chain reaction (cPCR) technique due to its simple mechanism, which requires no thermal cycling control. Fluorescence increment analysis and information entropy analysis are employed separately to determine whether the test samples contain target DNA (Positive) or not (Negative). The fluorescence increment method uses the brightness of the captured images before and after DNA amplification to calculate ΔF. ΔF values above a threshold level indicate that the test sample is positive. The information entropy method defines the probability, P(C/X), which indicates whether the fluorescence image tends towards a specific shape. If a DNA template is successfully amplified, the captured fluorescence image should be a perfect circle. P(C/X) provides a threshold of 0.5 to identify a circle and values above 0.5 indicate the test sample is positive. Experimental results show that P(C/X) is more effective than ΔF for determining DNA detection results. The information entropy analysis method is applied to ten mobile phones of three different brands equipped with camera sensors, which have pixel numbers ranging from 120 M to 800 M. The clinical evaluation study (n = 60) for screening hepatitis B virus (HBV) plasmid samples shows that the accuracy rate of all models of mobile phones ranges from 85% to 100%. This illustrates that successful DNA detection can be achieved using the most widely deployed electronic device.     

DNA computing using single-molecule hybridization detection

DNA computing aims at using nucleic acids for computing. Since micromolar DNA solutions can act as billions of parallel nanoprocessors, DNA computers can in theory solve optimization problems that require vast search spaces. However, the actual parallelism currently being achieved is at least a hundred million-fold lower than the number of DNA molecules used. This is due to the quantity of DNA molecules of one species that is required to produce a detectable output to the computations. In order to miniaturize the computation and considerably reduce the amount of DNA needed, we have combined DNA computing with single-molecule detection. Reliable hybridization detection was achieved at the level of single DNA molecules with fluorescence cross-correlation spectroscopy. To illustrate the use of this approach, we implemented a DNA-based computation and solved a 4-variable 4-clause instance of the computationally hard Satisfiability (SAT) problem.     

Unique invasions and resolutions: DNA repair proteins in meiotic recombination in Drosophila melanogaster

To ensure the accurate disjunction of homologous chromosomes during meiosis, most eukaryotes rely on physical connections called chiasmata, which form at sites of crossing over. In the absence of crossing over, homologs may segregate randomly, resulting in high frequencies of aneuploid gametes. The process of meiotic recombination poses unique problems for the cell that must be overcome to ensure normal disjunction of homologous chromosomes. How is it ensured that crossovers occur between homologous chromosomes, rather than between sister chromatids? What determines the number and location of crossovers? The functions of DNA repair proteins hold some of the answers to these questions. In this review, we discuss DNA repair proteins that function in meiotic recombination in Drosophila melanogaster. We emphasize the processes of strand invasion and Holliday junction resolution in order to shed light on the questions raised above. Also, we compare the variety of ways several eukaryotes perform these processes and the different proteins they require.     

Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage

Recombination between homologous DNA molecules is essential for the proper maintenance and duplication of the genome, and for the repair of exogenously induced DNA damage such as double-strand breaks. Homologous recombination requires the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DNA damage induction. We show that these foci are dynamic structures of which Rad51 is a stably associated core component, whereas Rad52 and Rad54 rapidly and reversibly interact with the structure. Furthermore, we show that the majority of the proteins are not part of the same multi-protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi-protein complexes, rather than stable holo-complexes, allows flexibility. In the case of DNA repair, for example, it will facilitate cross-talk between different DNA repair pathways and coupling to other DNA transactions, such as replication.     

The F-Box DNA helicase Fbh1 prevents Rhp51-dependent recombination without mediator proteins

A key step in homologous recombination is the loading of Rad51 onto single-stranded DNA to form a nucleoprotein filament that promotes homologous DNA pairing and strand exchange. Mediator proteins, such as Rad52 and Rad55-Rad57, are thought to aid filament assembly by overcoming an inhibitory effect of the single-stranded-DNA-binding protein replication protein A. Here we show that mediator proteins are also required to enable fission yeast Rad51 (called Rhp51) to function in the presence of the F-box DNA helicase Fbh1. In particular, we show that the critical function of Rad22 (an orthologue of Rad52) in promoting Rhp51-dependent recombination and DNA repair can be mostly circumvented by deleting fbh1. Similarly, the reduced growth/viability and DNA damage sensitivity of an fbh1(-) mutant are variously suppressed by deletion of any one of the mediators Rad22, Rhp55, and Swi5. From these data we propose that Rhp51 action is controlled through an interplay between Fbh1 and the mediator proteins. Colocalization of Fbh1 with Rhp51 damage-induced foci suggests that this interplay occurs at the sites of nucleoprotein filament assembly. Furthermore, analysis of different fbh1 mutant alleles suggests that both the F-box and helicase activities of Fbh1 contribute to controlling Rhp51.     

RecQ promotes toxic recombination in cells lacking recombination intermediate-removal proteins

The RecQ-helicase family is widespread, is highly conserved, and includes human orthologs that suppress genomic instability and cancer. In vivo, some RecQ homologs promote reduction of steady-state levels of bimolecular recombination intermediates (BRIs), which block chromosome segregation if not resolved. We find that, in vivo, E. coli RecQ can promote the opposite: the net accumulation of BRIs. We report that cells lacking Ruv and UvrD BRI-resolution and -prevention proteins die and display failed chromosome segregation attributable to accumulation of BRIs. Death and segregation failure require RecA and RecF strand exchange proteins. FISH data show that replication is completed during chromosome-segregation failure/death of ruv uvrD recA(Ts) cells. Surprisingly, RecQ (and RecJ) promotes this death. The data imply that RecQ promotes the net accumulation of BRIs in vivo, indicating a second paradigm for the in vivo effect of RecQ-like proteins. The E. coli RecQ paradigm may provide a useful model for some human RecQ homologs.     

Simultaneous detection of DNA-binding proteins using exo-Taq-based reaction

The DNA-binding protein (DBP) has a wide range of roles such as those in DNA repair, recombination, and gene expression. Recently, a microarray-based method has been developed for the high-throughput analysis of DNA-protein interactions. However, to maximize the advantages of this method, the detection process should be improved so that the method can be applied to many proteins without the use of antibody or sample labeling. Previously, we presented a primary report on the detection of DBP, which is applicable to the microarray format. The system consists of three steps: first, the target DBP in the sample solution is incubated with a probe DNA; second, the probe is digested with Exo (Exonuclease) III; finally, the probe is extended withTaq DNA polymerase using fluorescent dye-labeled dUTP as a substrate. The binding DBP protects the probe from digestion by Exo III. Therefore, only the DBP-bound probe allows the following extension. In this study, the simultaneous detection of multiple DBPs was examined, and then the DBPs were analyzed using a crude extract of the cultured cells to demonstrate the general applicability of the method. Our method can be applied to many DBPs using the same procedure and components, whereas in the antibody-based method, the same number of antibodies as DBPs is needed to detect target DBPs in ELISA (enzyme-linked immunosorbent assay). These results suggest that our method is useful for the high-throughput detection of DBPs in the microarray format.