#08 ATCG

Combined media monoprints on photographic paper. 8" x 10", DNA/Recombination Series. The Museum of Contemporary Photography, Chicago

#09 ACGT

Combined media monoprints on photographic paper. 8" x 10", DNA/Recombination Series. The Museum of Contemporary Photography, Chicago

#10 AGCT

Combined media monoprints on photographic paper. 8" x 10", DNA/Recombination Series. The Museum of Contemporary Photography, Chicago

#11 ATGC

Combined media monoprints on photographic paper. 8" x 10", DNA/Recombination Series. The Museum of Contemporary Photography, Chicago

#12 ACTG

Combined media monoprints on photographic paper. 8" x 10", DNA/Recombination Series. The Museum of Contemporary Photography, Chicago

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.

A tumour-derived mutant allele of XRCC2 preferentially suppresses homologous recombination at DNA replication forks

Homologous recombination repair (HRR) is required for both the repair of DNA double strand breaks (DSBs) and the maintenance of the integrity of DNA replication forks. To determine the effect of a mutant allele of the RAD51 paralog XRCC2 (342delT) found in an HRR-defective tumour cell line, 342delT was introduced into HRR proficient cells containing a recombination reporter substrate. In one set of transfectants, expression of 342delT conferred sensitivity to thymidine and mitomycin C and suppressed HRR induced at the recombination reporter by thymidine but not by DSBs. In a second set of transfectants, the expression of 342delT was accompanied by a decreased level of the full-length XRCC2. These cells were defective in the induction of HRR by either thymidine or DSBs. Thus 342delT suppresses recombination induced by thymidine in a dominant negative manner while recombination induced by DSBs appears to depend upon the level of XRCC2 as well as the expression of the mutant XRCC2 allele. These results suggest that HRR pathways responding to stalled replication forks or DSBs are genetically distinguishable. They further suggest a critical role for XRCC2 in HRR at replication forks, possibly in the loading of RAD51 onto gapped DNA.

Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

Cytosines, but Not Purines, Determine Recombination Activating Gene (RAG)-induced Breaks on Heteroduplex DNA Structures

The sequence specificity of the recombination activating gene (RAG) complex during V(D)J recombination has been well studied. RAGs can also act as structure-specific nuclease; however, little is known about the mechanism of its action. Here, we show that in addition to DNA structure, sequence dictates the pattern and efficiency of RAG cleavage on altered DNA structures. Cytosine nucleotides are preferentially nicked by RAGs when present at single-stranded regions of heteroduplex DNA. Although unpaired thymine nucleotides are also nicked, the efficiency is many fold weaker. Induction of single- or double-strand breaks by RAGs depends on the position of cytosines and whether it is present on one or both of the strands. Interestingly, RAGs are unable to induce breaks when adenine or guanine nucleotides are present at single-strand regions. The nucleotide present immediately next to the bubble sequence could also affect RAG cleavage. Hence, we propose “C(d)C(S)C(S)” (d, double-stranded; s, single-stranded) as a consensus sequence for RAG-induced breaks at single-/double-strand DNA transitions. Such a consensus sequence motif is useful for explaining RAG cleavage on other types of DNA structures described in the literature. Therefore, the mechanism of RAG cleavage described here could explain facets of chromosomal rearrangements specific to lymphoid tissues leading to genomic instability.

Evidence for the role of Mycobacteriumtuberculosis RecG helicase in DNA repair and recombination

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacteriumtuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M.tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichiacoli recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions.

Processing of DNA double-stranded breaks and intermediates of recombination and repair by saccharomyces cerevisiae Mre11 and its stimulation by Rad50, Xrs2, and Sae2 proteins

Saccharomyces cerevisiae RAD50, MRE11, and XRS2 genes are essential for telomere length maintenance, cell cycle checkpoint signaling, meiotic recombination, and DNA double-stranded break (DSB) repair via nonhomologous end joining and homologous recombination. The DSB repair pathways that draw upon Mre11-Rad50-Xrs2 subunits are complex, so their mechanistic features remain poorly understood. Moreover, the molecular basis of DSB end resection in yeast mre11-nuclease deficient mutants and Mre11 nuclease-independent activation of ATM in mammals remains unknown and adds a new dimension to many unanswered questions about the mechanism of DSB repair. Here, we demonstrate that S. cerevisiae Mre11 (ScMre11) exhibits higher binding affinity for single-over double-stranded DNA and intermediates of recombination and repair and catalyzes robust unwinding of substrates possessing a 3' single-stranded DNA overhang but not of 5' overhangs or blunt-ended DNA fragments. Additional evidence disclosed that ScMre11 nuclease activity is dispensable for its DNA binding and unwinding activity, thus uncovering the molecular basis underlying DSB end processing in mre11 nuclease deficient mutants. Significantly, Rad50, Xrs2, and Sae2 potentiate the DNA unwinding activity of Mre11, thus underscoring functional interaction among the components of DSB end repair machinery. Our results also show that ScMre11 by itself binds to DSB ends, then promotes end bridging of duplex DNA, and directly interacts with Sae2. We discuss the implications of these results in the context of an alternative mechanism for DSB end processing and the generation of single-stranded DNA for DNA repair and homologous recombination.

Investigating the role of apoptosis regulator EndoG on exogenous DNA uptake, stability, replication and recombination

Endonuclease G (EndoG) is a well conserved mitochondrial nuclease with dual lethal and vital roles in the cell. It non-specifically cleaves endogenous DNA following apoptosis induction, but is also active in non-apoptotic cells for mitochondrial DNA (mtDNA) replication and may also be important for replication, repair and recombination of genomic DNA. The aim of our study was to examine whether EndoG exerts similar activities on exogenous DNA substrates such as plasmid DNA (pDNA) and viral DNA vectors, considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA and nuclease activity in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four day time-course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus (a cationic liposome transfection reagent), targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances it may non-specifically cleave intracellular DNA regardless of its origin. To investigate possible effects of EndoG on viral DNA vectors, we constructed and evaluated AdsiEndoG, a first generation adenovirus (Ad5 ΔE1) vector encoding a shRNA directed against EndoG mRNA, along with appropriate Ad5 ΔE1 controls. Infection of HeLa cells with AdsiEndoG at a multiplicity of infection (MOI) of 10 p.f.u./cell resulted in an early cell proliferation defect, absent from cells infected at equivalent MOI with control Ad5 ΔE1 vectors. Replication of Ad5 ΔE1 DNA was detected for all vectors, but AdsiEndoG DNA accumulated to levels that were 50 fold higher than initially, four days after infection, compared to 14 fold for the next highest control Ad5 ΔE1 vector. Deregulation of the cell cycle by EndoG depletion, which is characterized by an accumulation of cells in the G2/M transition, is the most likely reason for the observed cell proliferation defect. The enhanced replication of AdsiEndoG is consistent with this conclusion, as Ad5 ΔE1 DNA replication is intimately related to cell cycling and prolongation or delay in G2/M greatly enhances this process. Furthermore, infection of HeLa with AdsiEndoG at MOI of 50 p.f.u./cell resulted in an almost complete disappearance of viable, adherent tumour cells from culture, whereas almost a third of the cells were still adherent after infection with control Ad5 ΔE1 vectors, relative to the non-infected control. Therefore, targeting of EndoG by RNAi is a viable strategy for improving the oncolytic properties of first generation adenovirus vectors. In addition, AdsiEndoG-mediated knockdown of EndoG reduced homologous recombination between pDNA substrates in HeLa cells. The effect was modest but, nevertheless demonstrated that the proposed role of EndoG in homologous recombination of cellular DNA also extends to exogenous DNA substrates.

Rare and fleeting: an example of interspecific recombination in animal mitochondrial DNA

Recombination is thought to occur only rarely in animal mitochondrial DNA ( mtDNA). However, detection of mtDNA recombination requires that cells become heteroplasmic through mutation, intramolecular recombination or ' leakage' of paternal mtDNA. Interspecific hybridization increases the probability of detecting mtDNA recombinants due to higher levels of sequence divergence and potentially higher levels of paternal leakage. During a study of historical variation in Atlantic salmon ( Salmo salar) mtDNA, an individual with a recombinant haplotype containing sequence from both Atlantic salmon and brown trout ( Salmo trutta) was detected. The individual was not an F1 hybrid but it did have an unusual nuclear genotype which suggested that it was a later-generation backcross. No other similar recombinant haplotype was found from the same population or three neighbouring Atlantic salmon populations in 717 individuals collected during 1948 - 2002. Interspecific recombination may increase mtDNA variability within species and can have implications for phylogenetic studies.

Detecting recombination in 4-taxa DNA sequence alignments with Bayesian hidden Markov models and Markov chain Monte Carlo

This article presents a statistical method for detecting recombination in DNA sequence alignments, which is based on combining two probabilistic graphical models: (1) a taxon graph (phylogenetic tree) representing the relationship between the taxa, and (2) a site graph (hidden Markov model) representing interactions between different sites in the DNA sequence alignments. We adopt a Bayesian approach and sample the parameters of the model from the posterior distribution with Markov chain Monte Carlo, using a Metropolis-Hastings and Gibbs-within-Gibbs scheme. The proposed method is tested on various synthetic and real-world DNA sequence alignments, and we compare its performance with the established detection methods RECPARS, PLATO, and TOPAL, as well as with two alternative parameter estimation schemes.