Are the SSB-Interacting Proteins RecO,RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion.     

In vivo recombination after chronic damage exposure falls to below spontaneous levels in "recombomice"

All forms of cancer are initiated by heritable changes in gene expression. Although point mutations have been studied extensively, much less is known about homologous recombination events, despite its role in causing sequence rearrangements that contribute to tumorigenesis. Although transgenic mice that permit detection of point mutations have provided a fundamental tool for studying point mutations in vivo, until recently, transgenic mice designed specifically to detect homologous recombination events in somatic tissues in vivo did not exist. We therefore created fluorescent yellow direct repeat mice, enabling automated detection of recombinant cells in vivo for the first time. Here, we show that an acute dose of ionizing radiation induces recombination in fluorescent yellow direct repeat mice, providing some of the first direct evidence that ionizing radiation induces homologous recombination in cutaneous tissues in vivo. In contrast, the same total dose of radiation given under chronic exposure conditions suppresses recombination to levels that are significantly below those of unexposed animals. In addition, global methylation is suppressed and key DNA repair proteins are induced in tissues from chronically irradiated animals (specifically AP endonuclease, polymerase beta, and Ku70). Thus, increased clearance of recombinogenic lesions may contribute to suppression of homologous recombination. Taken together, these studies show that fluorescent yellow direct repeat mice provide a rapid and powerful assay for studying the recombinogenic effects of both short-term and long-term exposure to DNA damage in vivo and reveal for the first time that exposure to ionizing radiation can have opposite effects on genomic stability depending on the duration of exposure.     

Use of green fluorescent protein/Flp recombinase fusion protein and flow cytometric sorting to enrich for cells undergoing Flp-mediated recombination

Flp recombinase has been used extensively for in vivo manipulation of eukaryotic DNA at specific sequences designated as FRT sites. We developed a method to use Flp-mediated recombination without the need for drug resistance or metabolic selection of cells in which recombination has occurred. We generated expression plasmids directing expression of fusion proteins consisting of Flp recombinase and green fluorescent protein (GFP) coding sequences. When the plasmids were introduced into K562 cells containing Flp recombinase substrates and transfected cells were selected for by flow cytometric sorting, GFP-positive cells were enriched 5- to 30-fold for Flp-mediated recombination events compared with unsorted cells. These studies demonstrate the usefulness of GFP/Flp recombinase fusion proteins to manipulate chromosomal DNA in vivo without requiring drug resistance or metabolic marker genes.     

Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins - Characterization and Use in Antiviral Screening

Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity). We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP) fused with the viral proteins IE-2, ppUL32 (pp150), and ppUL83 (pp65). In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI). The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus–infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture.     

DNA recombination: the replication connection.

Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.     

A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity

Accurate DNA double-strand break repair through homologous recombination is essential for preserving genome integrity. Disruption of the gene encoding RAD51, the protein that catalyzes DNA strand exchange during homologous recombination, results in lethality of mammalian cells. Proteins required for homologous recombination, also play an important role during DNA replication. To explore the role of RAD51 in DNA replication and DSB repair, we used a knock-in strategy to express a carboxy-terminal fusion of green fluorescent protein to mouse RAD51 (mRAD51-GFP) in mouse embryonic stem cells. Compared to wild-type cells, heterozygous mRad51^+/wt-GFP embryonic stem cells showed increased sensitivity to DNA damage induced by ionizing radiation and mitomycin C. Moreover, gene targeting was found to be severely impaired in mRad51^+/wt-GFP embryonic stem cells. Furthermore, we found that mRAD51-GFP foci were not stably associated with chromatin. From these experiments we conclude that this mRad51-GFP allele is an antimorphic allele. When this allele is present in a heterozygous condition over wild-type mRad51, embryonic stem cells are proficient in DNA replication but display defects in homologous recombination and DNA damage repair.     

Functional EGFP-dystrophin fusion proteins for gene therapy vector development

Transfection and transduction studies involving the use of the full-length dystrophin (11 kb) or the truncated mini-gene (6 kb) cDNAs are hampered by the large size of the resulting viral or non-viral expression vectors. This usually results in very low yields of transgene-expressing cells. Moreover, the detection of the few transgene-expressing cells is often tedious and costly. For these reasons, expression vectors containing the enhanced green fluorescent protein (EGFP) fused with the N-termini of mini- and full-length human dystrophin were constructed. These constructs were tested by transfection of Phoenix cells with Effectene, resulting after 48 h in a green fluorescent signal in 20% of cells. Analysis of the cell extracts by immunoblotting with the use of a monoclonal antibody specific to the dystrophin C-terminus confirmed the expression of EGFP-mini- (240 kDa) and EGFP-full-length human dystrophin (450 kDa) fusion proteins. Moreover, following the in vivo electroporation of the plasmids containing the EGFP-mini- and full-length dystrophin in mouse muscles, both fluorescent proteins were observed in cryostat sections in their normal location under the plasma membrane. This indicates that the fusion of EGFP to dystrophin or mini-dystrophin did not interfere with the normal localization of the protein. In conclusion, the fusion of EGFP provides a good tool for the search of the best methods to introduce mini- or full-length dystrophin cDNA in the cells (in vitro) or muscle fibers (in vivo) for the establishment of a treatment by gene therapy of Duchenne muscular dystrophy patients.     

Resolution of holliday junctions by RuvABC prevents dimer formation in rep mutants and UV-irradiated cells

In this work, we present evidence that indicates that RuvABC proteins resolve Holliday junctions in a way that prevents dimer formation in vivo. First, although arrested replication forks are rescued by recombinational repair in cells deficient for the Rep helicase, rep mutants do not require the XerCD proteins or the dif site for viability. This shows that the recombination events at arrested replication forks are generally not accompanied by the formation of chromosome dimers. Secondly, resolution of dimers into monomers is essential in the rep ruv strain because of an increased frequency of RecFOR recombination events in the chromosome of this mutant. This suggests that, in the absence of the Ruv proteins, chromosomal recombination leads to frequent dimerization. Thirdly, dif or xerC mutations increase the UV sensitivity of ruv-deficient cells 100-fold, whereas they do not confer UV sensitivity to ruv+ cells. This shows that recombinational repair of UV lesions is not accompanied by dimer formation provided that the RuvABC proteins are active. The requirement for dimer resolution in ruv strains is suppressed by the expression of the RusA Holliday junction resolvase; therefore, RusA also prevents dimer formation. We conclude that the inviability arising from a high frequency of dimer formation in rep or UV-irradiated cells is only observed in the absence of known enzymes that resolve Holliday junctions.     

Repair of DNA interstrand crosslinks may take place at the nuclear matrix

Host cell reactivation assay using Trioxsalen-crosslinked plasmid pEGFP-N1 showed that human cells were able to repair Trioxsalen interstrand crosslinks (ICL). To study the mechanism of this repair pathway, cells were transfected with the plasmids pEGFP-1, which did not contain the promoter of the egfp gene, and with pEGFP-G-, which did not contain the egfp gene. Neither of these plasmids alone was able to express the green fluorescent protein. After cotransfection with the two plasmids, 1%-2% of the cells developed fluorescent signal, which showed that recombination events had taken place in these cells to create DNA constructs containing the promoter and the gene properly aligned. When one or both of the plasmids were crosslinked with Trioxsalen, the recombination rate increased several fold. To identify the nuclear compartment where recombination takes place, cells were transfected with crosslinked pEGFP-N1 and the amount of plasmid DNA in the different nuclear fractions was determined. The results showed that Trioxsalen crosslinking increased the percentage of matrix attached plasmid DNA in a dose-dependent way. Immunoblotting experiments showed that after transfection with Trioxsalen crosslinked plasmids the homologous recombination protein Rad51 also associated with the nuclear matrix fraction. These studies provide a model system for investigating the precise molecular mechanisms that appear to couple repair of DNA ICL with nuclear matrix attachment.     

Mismatch repair proteins: key regulators of genetic recombination

Mismatch repair (MMR) systems are central to maintaining genome stability in prokaryotes and eukaryotes. MMR proteins play a fundamental role in avoiding mutations, primarily by removing misincorporation errors that occur during DNA replication. MMR proteins also act during genetic recombination in steps that include repairing mismatches in heteroduplex DNA, modulating meiotic crossover control, removing 3' non-homologous tails during double-strand break repair, and preventing recombination between divergent sequences. In this review we will, first, discuss roles for MMR proteins in repairing mismatches that occur during recombination, particularly during meiosis. We will also explore how studying this process has helped to refine models of double-strand break repair, and particularly to our understanding of gene conversion gradients. Second, we will examine the role of MMR proteins in repressing homeologous recombination, i.e. recombination between divergent sequences. We will also compare the requirements for MMR proteins in preventing homeologous recombination to the requirements for these proteins in mismatch repair.     

Replication-Dependent Sister Chromatid Recombination in Rad1 Mutants of Saccharomyces Cerevisiae

Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G(1) or the G(2) phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G(1), this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G(2) phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G(2). Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination.     

Applications of Fluorescence for Detecting Rare Sequence Rearrangements In Vivo

Homologous recombination (HR) is an important pathway for the accurate repair of potentially cytotoxic or mutagenic double strand breaks (DSBs), as well as double strand ends that arise due to replication fork breakdown. Thus, measuring HR events can provide information on conditions that induce DSB formation and replicative stress. To study HR events in vivo, we previously developed Fluorescent Yellow Direct Repeat (FYDR) mice in which a recombination event at an integrated transgene yields a fluorescent signal. Recently, we published an application of these mice demonstrating that fluorescent recombinant cells can be directly detected within intact pancreatic tissue. Here, we show that in situ imaging is a more sensitive method for detecting exposure-induced recombinant cells, yielding statistical significance with smaller cohorts. In addition, we show inter-mouse and gender-dependent variation in transgene expression, examine its impact on data interpretation, and discuss solutions to overcoming the effects of such variation. Finally, we also present data on EYFP expression, showing that several tissues, in addition to the pancreas, may be amenable for in situ detection of recombinant cells in the FYDR mice. The FYDR mice provide a unique tool for identifying genetic conditions and environmental exposures that induce genotoxic stress in a variety of tissues.     

Preferential post-replication repair of DNA lesions situated on the leading strand of plasmids in Escherichia coli

In Escherichia coli , RecF-dependent post-replication repair (PRR) permits cells to tolerate the potentially lethal effects of blocking lesions at the replication fork. We have developed an in vivo experimental system to study the PRR mechanisms that allow blocked replication forks to be rescued by homologous sequences. We show that approximately 80% of the PRR events observed in SOS-uninduced cells are generated by RecA-mediated excision repair, a novel nucleotide excision repair- and RecA/RecF-dependent mechanism, while 20% are generated by RecF-dependent homologous recombination. Moreover, we show that in a wild-type background, PRR is approximately an order of magnitude more efficient in processing DNA containing a blocked leading strand, as compared with a blocked lagging strand. This strand bias is abolished in cells that are deficient in nucleotide excision repair. These results are discussed in the context of recent models describing the mechanisms of replication past damaged templates.     

Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous DNA single-strand breaks

Homologous recombination is vital to repair fatal DNA damage during DNA replication. However, very little is known about the substrates or repair pathways for homologous recombination in mammalian cells. Here, we have compared the recombination products produced spontaneously with those produced following induction of DNA double-strand breaks (DSBs) with the I-SceI restriction endonuclease or after stalling or collapsing replication forks following treatment with thymidine or camptothecin, respectively. We show that each lesion produces different spectra of recombinants, suggesting differential use of homologous recombination pathways in repair of these lesions. The spontaneous spectrum most resembled the spectra produced at collapsed replication forks formed when a replication fork runs into camptothecin-stabilized DNA single-strand breaks (SSBs) within the topoisomerase I cleavage complex. We found that camptothecin-induced DSBs and the resulting recombination repair require replication, showing that a collapsed fork is the substrate for camptothecin-induced recombination. An SSB repair-defective cell line, EM9 with an XRCC1 mutation, has an increased number of spontaneous gammaH2Ax and RAD51 foci, suggesting that endogenous SSBs collapse replication forks, triggering recombination repair. Furthermore, we show that gammaH2Ax, DSBs, and RAD51 foci are synergistically induced in EM9 cells with camptothecin, suggesting that lack of SSB repair in EM9 causes more collapsed forks and more recombination repair. Furthermore, our results suggest that two-ended DSBs are rare substrates for spontaneous homologous recombination in a mammalian fibroblast cell line. Interestingly, all spectra showed evidence of multiple homologous recombination events in 8 to 16% of clones. However, there was no increase in homologous recombination genomewide in these clones nor were the events dependent on each other; rather, we suggest that a first homologous recombination event frequently triggers a second event at the same locus in mammalian cells.     

Rapid aggregate formation of the huntingtin N-terminal fragment carrying an expanded polyglutamine tract

Huntington's disease (HD) is caused by an expansion of the CAG repeat in the HD gene. The repeat is translated to the polyglutamine tract as huntingtin, the product of HD gene. Several studies showed that the expansion of polyglutamine tract leads to formation of cytoplasminc and/or intranuclear aggregates in vivo or in vitro. To understand the molecular mechanism of the aggregate formation, we studied the transient expression of HD exon 1-GFP fusion proteins in COS-7 cells. The fusion protein carrying 77 glutamine repeats aggregated in a time-dependent manner, while the fusion protein carrying 25 glutamine tract remained to be distributed diffusely in the cytoplasm even 72 hours after transfection. Initially, fluorescent signals were diffusely distributed in the COS-7 cells that were transfected with the construct containing the 77 CAG repeats. Approximately 40 hours later after the transfection, large aggregates grew very rapidly in those cells and the diffuse cytoplasmic fluorescence faded out. This process was completed within 40 minutes from the appearance of small aggregates in the perinuclear regions. The addition of cycloheximide reduced the frequencies of aggregate formation. A possibility was discussed that the aggregate formation was via nucleation. The focal concentration of mutated proteins in neurons may trigger the aggregate formation.     

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.     

Impaired Replication Stress Response in Cells from Immunodeficiency Patients Carrying Cernunnos/XLF Mutations

Non-Homologous End Joining (NHEJ) is one of the two major pathways of DNA Double Strand Breaks (DSBs) repair. Mutations in human NHEJ genes can lead to immunodeficiency due to its role in V(D)J recombination in the immune system. In addition, most patients carrying mutations in NHEJ genes display developmental anomalies which are likely the result of a general defect in repair of endogenously induced DSBs such as those arising during normal DNA replication. Cernunnos/XLF is a recently identified NHEJ gene which is mutated in immunodeficiency with microcephaly patients. Here we aimed to investigate whether Cernunnos/XLF mutations disrupt the ability of patient cells to respond to replication stress conditions. Our results demonstrate that Cernunnos/XLF mutated cells and cells downregulated for Cernunnos/XLF have increased sensitivity to conditions which perturb DNA replication. In addition, under replication stress, these cells exhibit impaired DSB repair and increased accumulation of cells in G2/M. Moreover Cernunnos/XLF mutated and down regulated cells display greater chromosomal instability, particularly at fragile sites, under replication stress conditions. These results provide evidence for the role of Cernunnos/XLF in repair of DSBs and maintenance of genomic stability under replication stress conditions. This is the first study of a NHEJ syndrome showing association with impaired cellular response to replication stress conditions. These findings may be related to the clinical features in these patients which are not due to the V(D)J recombination defect. Additionally, in light of the emerging important role of replication stress in the early stages of cancer development, our findings may provide a mechanism for the role of NHEJ in preventing tumorigenesis.     

Ataxin-1 fusion partners alter polyQ lethality and aggregation

Intranuclear inclusion bodies (IBs) are the histopathologic markers of multiple protein folding diseases. IB formation has been extensively studied using fluorescent fusion products of pathogenic polyglutamine (polyQ) expressing proteins. These studies have been informative in determining the cellular targets of expanded polyQ protein as well as the methods by which cells rid themselves of IBs. The experimental thrust has been to intervene in the process of polyQ aggregation in an attempt to alleviate cytotoxicity. However new data argues against the notion that polyQ aggregation and cytotoxicity are inextricably linked processes. We reasoned that changing the protein context of a disease causing polyQ protein could accelerate its precipitation as an IB, potentially reducing its cytotoxicity. Our experimental strategy simply exploited the fact that conjoined proteins influence each others folding and aggregation properties. We fused a full-length pathogenic ataxin-1 construct to fluorescent tags (GFP and DsRed1-E5) that exist at different oligomeric states. The spectral properties of the DsRed1-E5-ataxin-1 transfectants had the additional advantage of allowing us to correlate fluorochrome maturation with cytotoxicity. Each fusion protein expressed a distinct cytotoxicity and IB morphology. Flow cytometric analyses of transfectants expressing the greatest fluorescent signals revealed that the DsRed1-E5-ataxin-1 fusion was more toxic than GFP fused ataxin-1 (31.8+/-4.5% cell death versus 12.85+/-3%), although co-transfection with the GFP fusion inhibited maturation of the DsRed1-E5 fluorochrome and diminished the toxicity of the DsRed1-E5-ataxin-1 fusion. These data show that polyQ driven aggregation can be influenced by fusion partners to generate species with different toxic properties and provide new opportunities to study IB aggregation, maturation and lethality.     

A poliovirus minireplicon containing an inactive 2A proteinase is expressed in vaccinia virus-infected cells.

It has been difficult to evaluate the role of individual viral proteins in poliovirus replication because a suitable complementation system has not yet been developed. To approach this problem, we constructed a chimeric human immunodeficiency virus type 2 (HIV-2)-gag-poliovirus minireplicon in which regions of the gag gene of HIV-2 were inserted in the poliovirus genome between nucleotides 1174 and 2470. Transfection of this chimeric RNA into HeLa cells results in the replication of the minireplicon and expression of an HIV-2-gag-P1 fusion protein which can be immunoprecipitated with antibodies to HIV-2-gag. Expression of the HIV-2-gag-P1 fusion protein was dependent on replication of the chimeric RNA genome. Although the chimeric HIV-2-gag-poliovirus RNA genome replicated in poliovirus-infected cells, transfection of the chimeric HIV-2-gag-poliovirus genome into vaccinia virus-infected cells resulted in increased replication as measured by analysis of chimeric RNA. The increase in replication correlated with an increase in the expression of the HIV-2-gag-P1 fusion protein in vaccinia virus-infected cells. To characterize this system, we constructed a mutation in the 2A gene to change a cysteine at amino acid 109 to a serine. Expression of the HIV-2-gag-P1 fusion protein was not detected when the HIV-2-gag-poliovirus genome containing the 2A mutation was transfected into HeLa cells, demonstrating the mutation was lethal for replication. When the chimeric genome was transfected into poliovirus-infected cells, no RNA replication or expression of the HIV-2-gag-P1 fusion protein was observed. In contrast, transfection of this genome into vaccinia virus-infected cells resulted in replication of the chimeric RNA and expression of two proteins with larger molecular masses than the HIV-2-gag-P1 proteins, possibly representing HIV-2-gag-P1-2A and HIV-2-gag-P1-2ABC fusion proteins. The transfection of the chimeric HIV-2-gag-poliovirus genome containing the 2A mutation into poliovirus-vaccinia virus coinfected cells resulted in the expression and partial processing of the two larger HIV-2-gag-P1 fusion proteins to give the correct molecular mass for the HIV-2-gag-P1 fusion protein. The 2A mutation was reconstructed back into the full-length infectious cDNA of poliovirus. Transfection of this cDNA into vaccinia virus-infected cells followed by immunoprecipitation with anticapsid antibodies demonstrated the presence of two proteins with molecular masses larger than P1, possibly P1-2A and P1-2ABC fusion proteins.(ABSTRACT TRUNCATED AT 400 WORDS)