Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo

Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.DNA double-strand break repair (DSBR) is essential for cell survival and repair-deficient cells are highly sensitive to chromosome breakage. In Escherichia coli, a single unrepaired DNA DSB per replication cycle is lethal, illustrating the critical nature of the repair reaction (1). DSBR in E. coli is mediated by homologous recombination, which relies on the RecA protein to efficiently recognize DNA sequence identity between two molecules. RecA homologs are widely conserved from bacteriophages to mammals, where they are known as the Rad51 proteins (2). The RecA protein plays its central role by binding single-stranded DNA (ssDNA) to form a presynaptic filament that searches for a homologous double-stranded DNA (dsDNA) donor from which to repair. It then catalyzes a strand-exchange reaction to form a joint molecule (3), which is stabilized by the branch migration activities of the RecG and RuvAB proteins (4). The joint molecule is then resolved by cleavage at its four-way Holliday junction by the nuclease activity of RuvABC (5, 6).RecA binding at the site of a DSB is dependent on the activity of the RecBCD enzyme (Fig. 1A). RecBCD is a helicase-nuclease that binds to dsDNA ends, then separates and unwinds the two DNA strands using the helicase activities of the RecB and RecD subunits (see refs. 7 and 8 for recent reviews). RecD is the faster motor of the two and this consequently results in the formation of a ssDNA loop ahead of RecB (Loop 1 in Fig. 1A) (9). As the enzyme translocates along dsDNA, the 3′-terminated strand is continually passed through the Chi-scanning site thought to be located in the RecC protein (10). When a Chi sequence (the octamer 5′-GCTGGTGG-3′) enters this recognition domain, the RecD motor is disengaged and the 3′ strand continues to be unwound by RecB. Under in vitro conditions, where the concentration of magnesium exceeds that of ATP, the 3′ end (unwound by RecB) is rapidly digested before Chi recognition, whereas the 5′ end (unwound by RecD) is intermittently cleaved (11, 12). After Chi recognition the 3′ end is no longer cleaved but the nuclease domain of RecB continues to degrade the 5′ end as it exits the enzyme (11, 12). Under in vitro conditions where the concentration of ATP exceeds that of magnesium, unwinding takes place but the only site of cleavage detected is ∼5 nucleotides 3′ of the Chi sequence (13, 14). Because the RecB motor continues to operate while the RecD motor is disengaged, Loop 1 is converted to a second loop located between the RecB and RecC subunits or to a tail upon release of the Chi sequence from its recognition site. We therefore describe this single-stranded region as Loop/Tail 2 in Fig. 1A. After the whole of Loop 1 is converted to Loop/Tail 2, this second single-stranded region continues to grow as long as the RecB subunit unwinds the dsDNA. The RecBCD enzyme enables RecA protein to load on to Loop/Tail 2 to generate the presynaptic filament necessary to search for homology and initiate strand-exchange (15). Finally, the RecBCD enzyme stops translocation and disassembles as it dissociates from the DNA, releasing a DNA-free RecC subunit (16).Open in a separate windowFig. 1.DSBR in E. coli. (A and B) Schematic representation of DSB processing by the RecBCD complex. (A) Before Chi recognition, both the RecB and RecD motors progress along the DNA. RecD is the faster motor and as a result a loop of ssDNA (Loop 1) is formed ahead of the slower RecB motor. The 3′ ssDNA strand is scanned for the Chi sequence by the RecC protein. (B) After Chi recognition, RecBCD likely undergoes a conformational change so that only the RecB motor is engaged. The RecA protein is recruited by the RecB nuclease domain and loaded onto the ssDNA loop generated by RecB unwinding to promote RecA nucleoprotein filament formation. In this schematic representation, the Chi site is shown held in its recognition site. However, the Chi site will be released either by disassembly of the RecBCD complex or at some point before this and the second single-stranded region on the 3′ terminating strand will be converted from a loop to a tail. Therefore, this region is denoted Loop/Tail 2. The mathematical model described in SI Appendix does not depend on the ATP/magnesium dependent differential cleavage of DNA strands (7, 8), nor does it depend on the precise time that the 3′ end is released from the complex following Chi recognition. (C) The hairpin endonuclease SbcCD is used to cleave a 246-bp interrupted palindrome inserted in the lacZ gene of the E. coli chromosome. Cleavage of this DNA hairpin results in the generation of a site-specific DSB on only one of a pair of replicating sister chromosomes, thus leaving an intact sister chromosome to serve as a template for repair by homologous recombination.Our understanding of the action of RecBCD and RecA has been the result of more than 40 years of genetic analysis and biochemical investigation of these purified proteins in vitro. However, relatively little is known about their activities on the genomic scale. To investigate these reactions in vivo, we have used RecA chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) in an experimental system that allows us to introduce a single and fully repairable DSB into the chromosome of E. coli (1). Because DSBR by homologous recombination normally involves the repair of a broken chromosome by copying the information on an unbroken sister chromosome, our laboratory has previously developed a procedure for the cleavage of only one copy of two genetically identical sister chromosomes (1). We have made use of the observation that the hairpin nuclease SbcCD specifically cleaves only one of the two sister chromosomes following DNA replication through a 246-bp interrupted palindrome to generate a two-ended DSB (1). As shown in Fig. 1B, this break is fully repairable and we have shown that recombination-proficient cells suffer very little loss of fitness in repairing such breaks (17).Here we investigate in vivo and in a quantitative manner the first steps of DSBR: because the outcome of RecBCD action is understood to be the loading of RecA on DNA in a Chi-dependent manner, we use RecA-ChIP to reveal the consequences of RecBCD action on a genomic scale during DSBR. Analyses of most ChIP-Seq datasets focus on the identification of regions of significant enrichment of a given protein but do not take into account the underlying mechanisms giving rise to the binding (18). We reasoned that given the detailed mechanistic understanding of RecBCD in vitro, we could gain a deeper insight into its in vivo functions by developing a mathematical model of RecBCD action that would enable us to estimate the mechanistic parameters of the complex in live cells. Our ChIP data indicate that RecA is indeed loaded on to DNA in a Chi-dependent manner and we have used our mathematical model to infer the parameters of RecBCD action in vivo on a genomic scale. Furthermore, our analysis reveals that DSBR at lacZ induces DSBR in the terminus region of the chromosome, an unanticipated observation illuminated by the genomic scale of our data.     

Frequent spontaneous sister chromatid exchange in hepatocytes of transgenic mice harboring the SV40-T antigen gene

Summary In order to shed light on the causal mechanisms of hepatocarcinogenesis in the transgenic mouse into which the albumin-promotor-regulated SV40-T antigen gene has been introduced (T⁺ mouse), and especially on the frequent chromosomal aberrations seen in cultured hepatocytes and hepatocellular neoplasms derived from such animals, the frequency of sister chromatid exchange (SCE) and karyotype abnormalities were investigated in a hepatocyte primary culture system. Cells were obtained through collagenase perfusion from T⁺ mice at 16–18 days of age, when no morphological changes are apparent, and from nontransgenic littermates, and cultured in the presence of bromodeoxyuridine. SCE was seen in transgenic hepatocytes twice as frequently as in their normal counterparts. No karyotype abnormalities in terms of numerical change or gross aberration were detected at this phase. The results thus suggest mutagenic properties for the T antigen, which may play an important role in hepatocarcinogenesis in this transgenic mouse.Abbreviations SV40 simian virus 40 - T antigen, tumor antigen - SCE sister chromatid exchange - T mouse, transgenic mouse in which the SV40-T antigen gene has been introduced - HCC hepatocellular carcinoma - BrdU bromodeoxyuridine     

Meiotic instability of CAG repeat tracts occurs by double-strand break repair in yeast

Expansion of trinucleotide repeats is associated with a growing number of human diseases. The mechanism and timing of expansion of the repeat tract are poorly understood. In humans, trinucleotide repeats show extreme meiotic instability, and expansion of the repeat tract has been suggested to occur in the germ-line mitotic divisions or postmeiotically during early divisions of the embryo. Studies in model organisms have indicated that polymerase slippage plays a major role in the repeat tract instability and meiotic instability is severalfold higher than the mitotic instability. We show here that meiotic instability of the CAG/CTG repeat tract in yeast is associated with double-strand break (DSB) formation within the repeated sequences, and that the DSB formation is dependent on the meiotic recombination machinery. The DSB repair results in both expansions and contractions of the CAG repeat tract.     

Endogenous DNA double-strand breaks: production, fidelity of repair, and induction of cancer

This article extends our previous quantitative analysis of the relationship between the dynamics of the primary structure of DNA and mutagenesis associated with single-strand lesions to an analysis of the production and processing of endogenous double-strand breaks (EDSBs) and to their implications for oncogenesis. We estimate that in normal human cells approximately 1% of single-strand lesions are converted to approximately 50 EDSBs per cell per cell cycle. This number is similar to that for EDSBs produced by 1.5-2.0 Gy of sparsely ionizing radiation. Although EDSBs are usually repaired with high fidelity, errors in their repair contribute significantly to the rate of cancer in humans. The doubling dose for induced DSBs is similar to doubling doses for mutation and for the induction of carcinomas by ionizing radiation. We conclude that rates of production of EDSBs and of ensuing spontaneous mitotic recombination events can account for a substantial fraction of the earliest oncogenic events in human carcinomas.     

High sister chromatid exchange among a sample of traffic policemen in Bangkok, Thailand

There are several volatile substances from the traffic, including benzene, toluene, carbon monoxide, lead and formaldehyde. Most of these substances are considered carcinogens. Police are at occupational risk for toxic fume exposure. This study compared sister chromatid exchange (SCE), a marker for genotoxicity, among a sample of Thai traffic policemen in Bangkok with healthy control subjects. Thirty police officers (all male) and 20 controls were included in this study. The average (mean+/-SD) SCE for policemen and controls were 4.40+/-0.93/cell and 0.24+/-0.12/cell, respectively. A significantly higher SCE among the policemen was observed. Concern for and prevention of toxic substance exposure in traffic police officers should be made a national goal.     

Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

Chloroplast DNA (cpDNA) is under great photooxidative stress, yet its evolution is very conservative compared with nuclear or mitochondrial genomes. It can be expected that DNA repair mechanisms play important roles in cpDNA survival and evolution, but they are poorly understood. To gain insight into how the most severe form of DNA damage, a double-strand break (DSB), is repaired, we have developed an inducible system in Arabidopsis that employs a psbA intron endonuclease from Chlamydomonas, I-CreII, that is targeted to the chloroplast using the rbcS1 transit peptide. In Chlamydomonas, an I-CreII-induced DSB in psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20–60 bp) abundant in that genome; Arabidopsis cpDNA is very repeat poor, however. Phenotypically strong and weak transgenic lines were examined and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of DNA at the psbA locus, but not cpDNA as a whole, in the strongly expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site indicative of DSB repair using microhomology (6–12 bp perfect repeats, or 10–16 bp with mismatches) and no homology. These results provide evidence of alternative DSB repair pathways in the Arabidopsis chloroplast that resemble the nuclear, microhomology-mediated and nonhomologous end joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, the data suggest an evolutionary relationship may exist between the repeat structure of the genome and the organelle''s ability to repair broken chromosomes.     

Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination.

Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms. We studied the repair of genomic DSBs by homologous sequences in plants. Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme. We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude. Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways. In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast. Homologous recombination of the minor pathway is restricted to one side of the DSB as described by the nonconservative one-sided invasion model. The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process. The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation.     

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.Chromosomal double-strand breaks (DSBs), considered the most cytotoxic form of DNA damage, occur as a result of normal cellular processes (1, 2) as well as cancer therapies (3–5). The cellular DNA damage response (DDR) and repair pathways responsible for maintaining genomic integrity are highly regulated and synchronized processes, both temporally and spatially, involving the coordinated recruitment, assembly, and disassembly of numerous macromolecular complexes (6, 7). In mammalian cells, nonhomologous end-joining (NHEJ) is the primary DSB repair pathway; it is active throughout the cell cycle and is crucial for viability. Dysfunctional NHEJ is associated with several clinical conditions, including LIG4 syndrome and severe combined immunodeficiency (1, 8). Despite its importance, however, the details of how the NHEJ complex assembles at DSBs, brings together a pair of breaks, and organizes subsequent catalytic repair steps remain unknown.In NHEJ, DSBs are initially recognized by the Ku 70/80 heterodimer (Ku), which encircles dsDNA ends (Ku:DNA) and serves as a molecular scaffold for recruitment of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4 like factor), and DNA ligase IV (LigIV) (1, 9–14). Previous NHEJ models suggested that after binding of Ku to DNA ends, DNA-PKcs binds Ku:DNA to form a DNA-PK holoenzyme and bridges the broken ends (15–18); however, recent experiments indicate that DNA-PKcs may have different roles in NHEJ, such as the stabilization of core NHEJ factors, recruitment and retention of accessory factors, involvement in the DDR signaling cascade, and repair of complex and clustered DSBs (19–25). In addition, recent structural studies have shown that XRCC4 and XLF form filamentous structures in vitro (26–28). Whether such filaments mediate repair in vivo has not yet been determined.Our present understanding of the cellular NHEJ response to DSBs is based primarily on in vitro biochemical and structural studies done with purified proteins, together with cellular observations in which a radiologic or pharmacologic stimulus damages DNA, allowing observation of the repair process (29–31). Typically, cellular assays rely on a microscope to read out the response, looking for colocalization of DSB repair proteins with large DDR foci; however, conventional microscopy methods allow for only an inferential approach, given that the resolution limit of light is two orders of magnitude greater than the length scale of proteins. Here we used super-resolution (SR) localization microscopy and single-molecule FRET (smFRET) to analyze the cellular organization of NHEJ proteins and define the dynamics associated with end joining in vitro. SR microscopy circumvents the conventional resolution limit of light microscopy by temporally separating emitting fluorophores and computationally fitting the location of each below the diffraction limit. Reconstructing thousands of points in this manner generates an image with a resolution typically an order of magnitude better that that of conventional microscopy (32–34). In addition to this approach, we used smFRET, a powerful method capable of monitoring the dynamics of individual nucleoprotein complexes in real time (35).Using SR microscopy, we identified previously uncharacterized repair intermediates formed at DSBs in which Ku resides at the break site and XRCC4, XLF, and LigIV form long filamentous structures around and over DSB sites. We categorized these intermediates into two different structural subtypes, and defined their kinetics and the structural transitions that occur during the progression of repair. We further verified the formation of these structures using SR imaging analysis of NHEJ reactions carried out in vitro with recombinant proteins. Finally, we used smFRET to characterize the dynamics of end-joining in vitro, revealing that XRCC4/XLF/LigIV mediates end synapsis, and that after initial pairing, the DNA ends undergo dynamic interactions. Our findings identify XRCC4/XLF/LigIV filaments forming on either side of the break and then merging as a key step in DSB repair via NHEJ, and provide a detailed mechanism that is radically different from current models of NHEJ.     

Cytogenetic effects of Me-CCNU on sister chromatid exchange frequency, cellular kinetics and chromosome aberrations

1-(2-Chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (Me-CCNU) was tested for its in vitro effects on sister chromatid exchanges (SCE), cellular kinetics and chromosome aberrations in CHO cells. There was a relationship between the inhibitory activity of the drug and the cytogenetic damage, which was dose dependent. Increase in SCE values were highly significant (p less than 0.001) for all the four concentrations used. It also delayed the cell cycle progression. Inhibition of DNA synthesis results in increased frequency of chromosomal aberrations, which was highly significant for the higher concentrations, i.e. 5 micrograms and 10 micrograms Me-CCNU/ml.     

Replication and homologous recombination repair regulate DNA double-strand break formation by the antitumor alkylator ecteinascidin 743

Adducts induced by the antitumor alkylator ecteinascidin 743 (ET-743, Yondelis, trabectedin) represent a unique challenge to the DNA repair machinery because no pathway examined to date is able to remove the ET adducts, whereas cells deficient in nucleotide excision repair show increased resistance. We here describe the processing of the initial ET adducts into cytotoxic lesions and characterize the influence of cellular repair pathways on this process. Our findings show that exposure of proliferating mammalian cells to pharmacologically relevant concentrations of ET-743 is accompanied by rapid formation of DNA double-strand breaks (DSBs), as shown by the neutral comet assay and induction of focalized phosphorylated H2AX. The ET adducts are stable and can be converted into DSBs hours after the drug has been removed. Loss of homologous recombination repair has no influence on the initial levels of DSBs but is associated with the persistence of unrepaired DSBs after ET-743 is removed, resulting in extensive chromosomal abnormalities and pronounced sensitivity to the drug. In comparison, loss of nonhomologous end-joining had only modest effect on the sensitivity. The identification of DSB formation as a key step in the processing of ET-743 lesions represents a novel mechanism of action for the drug that is in agreement with its unusual potency. Because loss of repair proteins is common in human tumors, expression levels of selected repair factors may be useful in identifying patients particularly likely to benefit, or not, from treatment with ET-743.     

Bisphenol A impairs the double-strand break repair machinery in the germline and causes chromosome abnormalities

Bisphenol A (BPA) is a highly prevalent constituent of plastics that has been associated with diabetes, cardiovascular disease, and an increased risk of miscarriages in humans. In mice, BPA exposure disrupts the process of meiosis; however, analysis of the affected molecular pathways is lagging and has been particularly challenging. Here we show that exposure of the nematode Caenorhabditis elegans to BPA, at internal concentrations consistent with mammalian models, causes increased sterility and embryonic lethality. BPA exposure results in impaired chromosome synapsis and disruption of meiotic double-strand break repair (DSBR) progression. BPA carries an anti-estrogenic activity in the germline and results in germline-specific down-regulation of DSBR genes, thereby impairing maintenance of genomic integrity during meiosis. C. elegans therefore constitutes a model of remarkable relevance to mammals with which to assess how our chemical landscape affects germ cells and meiosis.     

Double-strand break repair by homologous recombination in primary mouse somatic cells requires BRCA1 but not the ATM kinase

Homology-directed repair (HDR) is a critical pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. Efficient HDR is thought to be crucial for maintenance of genomic integrity during organismal development and tumor suppression. However, most mammalian HDR studies have focused on transformed and immortalized cell lines. We report here the generation of a Direct Repeat (DR)-GFP reporter-based mouse model to study HDR in primary cell types derived from diverse lineages. Embryonic and adult fibroblasts from these mice as well as cells derived from mammary epithelium, ovary, and neonatal brain were observed to undergo HDR at I-SceI endonuclease-induced DSBs at similar frequencies. When the DR-GFP reporter was crossed into mice carrying a hypomorphic mutation in the breast cancer susceptibility gene Brca1, a significant reduction in HDR was detected, showing that BRCA1 is critical for HDR in somatic cell types. Consistent with an HDR defect, Brca1 mutant mice are highly sensitive to the cross-linking agent mitomycin C. By contrast, loss of the DSB signaling ataxia telangiectasia-mutated (ATM) kinase did not significantly alter HDR levels, indicating that ATM is dispensable for HDR. Notably, chemical inhibition of ATM interfered with HDR. The DR-GFP mouse provides a powerful tool for dissecting the genetic requirements of HDR in a diverse array of somatic cell types in a normal, nontransformed cellular milieu.DNA damage poses a threat to genomic integrity and must be repaired in an accurate and timely manner for the health and survival of the organism. A particularly cytotoxic lesion is a chromosomal double-strand break (DSB), which can arise from endogenous sources, including DNA replication and antigen receptor rearrangements in lymphocytes, as well as exogenous sources, such as ionizing radiation (IR) (1, 2). DSBs activate an elaborate cellular signaling network of proteins, a key component of which is the ataxia telangiectasia-mutated (ATM) protein kinase (3, 4). There are three major pathways for repairing DSBs in mammalian cells: (i) homology-directed repair (HDR), (ii) nonhomologous end joining (NHEJ), and (iii) single-strand annealing (SSA) (2, 5, 6). HDR, which is considered the most precise of the three repair pathways, uses a homologous donor sequence as a template for the repair event. Template preference is biased to the sister chromatid in mammalian cells, thus restoring the original sequence before damage. NHEJ refers to joining of ends without the use of extensive homology, and it is often accompanied by modification of the sequence surrounding the break site (1). SSA occurs when complementary strands from sequence repeats flanking the DSB anneal to each other, resulting in repair of the DSB but deletion of the intervening sequence (5). Consequently, both NHEJ and SSA are considered to be more error-prone than HDR. The importance of HDR to the organism is emphasized by the requirement for HDR factors for development, tumor suppression, and fertility (5).HDR was established as a DSB repair pathway in mammalian cells using genomic reporters into which a DSB is introduced by the I-SceI endonuclease (7–9). Repair of the DSB from the homologous template on the sister chromatid or the same chromatid leads to expression of a scoreable marker, such as GFP from the DR-GFP reporter (10). Use of such reporters has led to direct evidence for the role of several proteins in HDR, including the breast cancer suppressors BRCA1 and BRCA2 (11, 12). However, these studies have been performed in transformed cell lines and immortalized ES cells, raising questions as to their relevance to normal somatic cells. Some studies have suggested that DSB repair pathways are developmentally regulated, with HDR being critical during embryonic cell cycles and NHEJ in differentiated tissues (13). Furthermore, a critical protein in HDR, the RAD51 recombinase, has been reported to be overexpressed in tumor cell lines, suggesting that HDR levels are altered in transformed cells (14, 15).In the present study, we describe a mouse model to analyze the efficiency of HDR in primary somatic cell types. To this end, the DR-GFP reporter was targeted to a defined chromosomal locus in the mouse genome. We found that primary somatic cell types derived from different lineages from DR-GFP mice, including fibroblasts, brain (glial) cells, and mammary epithelial cells, undergo HDR at I-SceI–induced DSBs at similar frequencies. The frequency of HDR is lower in the tested somatic cell types compared with ES cells, which correlates with the lower fraction of cells in S phase in the more differentiated cell types. To examine the genetic requirements for HDR in somatic cells, we crossed the DR-GFP reporter into mice carrying mutations in the Brca1 and Atm genes. We found that BRCA1 is necessary for efficient HDR in somatic cells. By contrast, ATM is not required for HDR in primary fibroblasts, although chemical inhibition of ATM can interfere with HDR.     

Induction by alkylating agents of sister chromatid exchanges and chromatid breaks in Fanconi's anemia.

Sister chromatid exchanges, which may reflect chromosome repair in response to certain types of DNA damage, provide a means of investigating the increased chromosome fragility characteristic of Fanconi's anemia. By a recently developed technique using 33258 Hoechst and 5-bromodeoxyuridine, it was observed that the baseline frequency of sister chromatid exchanges in phytohemagglutinin-stimulated lymphocytes from four males with Fanconi's anemia differed little from that of normal lymphocytes. However, addition of the bifunctional alkylating agent mitomycin C (0.01 or 0.03 mug/ml) to the Fanconi's anemia cells during culture induces less than half of the increase in exchanges found in identically treated normal lymphocytes. This reduced increment in exchanges in accompanied by a partial suppression of mitosis and a marked increase in chromatid breaks and rearrangements. Many of these events occur at sites of incomplete chromatid interchange. The increase in sister chromatid exchanges induced in Fanconi's anemia lymphocytes by the monofunctional alkylating agent ethylmethane sulfonate (0.25 mg/ml) was slightly less than that in normal cells. Lymphocytes from two sets of parents of the patients with Fanconi's anemia exhibited a normal response to alkylating agents, while dermal fibroblasts from two different patients with Fanconi's anemia reacted to mitomycin C with an increase in chromatid breaks, but a nearly normal increment of sister chromatid exchanges. The results suggest that chromosomal breaks and rearrangements in Fanconi's anemia lymphocytes may result from a defect in a form of repair of DNA damage.     

Association of sister chromatid exchange frequencies in patients with ankylosing spondylitis with and without HLA-B27

BACKGROUND: The analysis of sister chromatid exchange (SCE) is a cytogenetic technique used to show DNA damage due to an exchange of DNA fragments between sister chromatids. OBJECTIVE: To determine whether HLA-B27 positive patients with ankylosing spondylitis (AS) were associated with higher SCE frequencies than patients without B27. METHODS: Lymphocytes from 38 patients with AS (15 women, 23 men) and 34 control subjects were examined. Peripheral lymphocytes were cultured in darkness for 72 hours in BrdU added culture. Metaphase chromosomes were stained with a fluorescence and a Giemsa stain after a standard harvest procedure. RESULTS: The frequency of SCE was significantly increased in patients with AS compared with controls (p<0.001). Furthermore, the SCE frequencies in patients with positive HLA-B27 was much higher than in patients with negative HLA-B27 (p<0.001). The difference between SCE frequencies in the control groups with and without HLA-B27 was not significant. CONCLUSION: There is a strong association between HLA-B27 and the frequencies of SCE in patients with AS.     

Effect of tumor promoters, protease inhibitors, and repair processes on x-ray-induced sister chromatid exchanges in mouse cells.

The induction of sister chromatid exchanges (SCE) in the second postirradiation mitosis was studied in mouse 10T1/2 cells irradiated with 400 rads (4 grays) and maintained in stationary growth for several hours after x-ray exposure (similar to liquid holding recovery experiments in bacterial cells). X-irradiation with no recovery period induced few SCE. With short recovery intervals, however, the SCE frequency rose in parallel with the increase in survival, reaching a maximum increase of 2-fold after 4 hr; SCE declined with longer recovery intervals. The influence of postirradiation incubation with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and with the protease inhibitors antipain and leupeptin was studied on spontaneous, x-ray-induced (no recovery), and recovery-induced (4 hr) SCE. TPA (0.1 microgram/ml and 1.0 microgram/ml) increased the frequency of both spontaneous and direct x-ray-induced SCE, but not of recovery-induced SCE. Incubation with the protease inhibitors suppressed both TPA- and recovery-induced SCE, but had no effect on direct x-ray-induced SCE. These results are discussed in relation to the hypothesis that promotional events in carcinogenesis may involve the expression of mutational damage in cells by mitotic segregation.     

Progesterone, but not estrogen, stimulates vessel maturation in the mouse endometrium

The human endometrium undergoes regular periods of growth and regression, including concomitant changes in the vasculature, and is one of the few adult tissues where significant angiogenesis and vascular maturation occurs on a routine, physiological basis. The aim of this study was to investigate the effects of estrogen and progesterone on endometrial vascular maturation in mice. Endometrial tissues were collected from early pregnant mice (d 1-4) and ovariectomized mice given a single 17beta-estradiol (100 ng) injection 24 h before dissection (short-term estrogen regime) or three consecutive daily injections of progesterone (1 mg) with/without estrogen priming (progesterone regime). Experiments were then repeated with the inclusion of mice treated concurrently with progesterone and either RU486 or a vascular endothelial growth factor-A antiserum. Proliferating vascular mural cells (PVMC) were observed on d 3-4 of pregnancy, corresponding with an increase in circulating progesterone. A significant increase in PVMC and alpha-smooth muscle actin (labels mural cells) coverage of vessel profiles were observed in mice treated with progesterone in comparison to controls; no significant change was noted in mice treated with estrogen or with vascular endothelial growth factor antiserum. RU486 treatment did not inhibit the progesterone-induced increases in PVMC and mural cell coverage, although progesterone-induced changes in endothelial and epithelial cell proliferation were inhibited. These results show that progesterone, but not estrogen, stimulates vessel maturation in the mouse endometrium. The work illustrates the relevancy of the mouse model for understanding endometrial vascular remodeling during the menstrual cycle and in response to the clinically important progesterone receptor antagonist RU486.     

Natural insertions in rice commonly form tandem duplications indicative of patch-mediated double-strand break induction and repair

The insertion of DNA into a genome can result in the duplication and dispersal of functional sequences through the genome. In addition, a deeper understanding of insertion mechanisms will inform methods of genetic engineering and plant transformation. Exploiting structural variations in numerous rice accessions, we have inferred and analyzed intermediate length (10–1,000 bp) insertions in plants. Insertions in this size class were found to be approximately equal in frequency to deletions, and compound insertion–deletions comprised only 0.1% of all events. Our findings indicate that, as observed in humans, tandem or partially tandem duplications are the dominant form of insertion (48%), although short duplications from ectopic donors account for a sizable fraction of insertions in rice (38%). Many nontandem duplications contain insertions from nearby DNA (within 200 bp) and can contain multiple donor sources—some distant—in single events. Although replication slippage is a plausible explanation for tandem duplications, the end homology required in such a model is most often absent and rarely is >5 bp. However, end homology is commonly longer than expected by chance. Such findings lead us to favor a model of patch-mediated double-strand-break creation followed by nonhomologous end-joining. Additionally, a striking bias toward 31-bp partially tandem duplications suggests that errors in nucleotide excision repair may be resolved via a similar, but distinct, pathway. In summary, the analysis of recent insertions in rice suggests multiple underappreciated causes of structural variation in eukaryotes.Genomic DNA insertion causes genome expansion and, potentially, the rearrangement and diffusion of protein domains and regulatory elements throughout the genome (1, 2). Additionally, genetic engineers generally aim to integrate specific DNA into the nuclear genome, so the natural mechanisms by which this integration occurs may serve as a starting point to elaborate and improve genome modification (3, 4). Common causes of gene-sized insertions are unequal recombination (5), transposable element replication (1), and ectopic recombination stimulated by double-strand breaks (DSBs) in the genome (2, 6). Shorter events are less well characterized, but it appears that these can be created by similar processes (7). Still, high-throughput sequencing of DSB repair events in humans (8) and plants (9) suggests that insertions related to induced breaks are very rare and very short.Although the processes described above can produce duplications at distant genetic loci, the most common form of non-microsatellite-associated insertions in humans is tandem duplications (10). Once created, tandem duplications can be dramatically expanded by unequal recombination or replication slippage. Such duplications may be deleterious, or they may be promoted by selection for a novel or expanded function (11, 12).Although tandem repeats are ubiquitous in eukaryotic genomes, the mechanisms for their origin are still in question. Early analysis of human indel mutations indicated that replication slippage was the most effective model to explain the origin of assorted repeats (13). In other studies, longer, de novo tandem duplications were also hypothesized to be caused by slippage because, out of 85 insertions producing such duplications, 50 were associated with flanking repeats >2 bp (14). Replication slippage would presumably require a preexisting short repeat because priming must occur between the end of the loop that will become the duplication and the position to where replication slips. Authors of more recent work investigating insertions across the human genome suggest alternatives to replication slippage on the grounds that homology is often either nonexistent or very short, whereas the length of homology and the length of insertion are not correlated (10). These researchers favor a model based on DSBs being repaired by nonhomologous end-joining (NHEJ). However, conventional models of DSB repair are strained to predict tandem duplications >10 bp, much less >100 bp. Such models require extensive single-stranded, complementary ends to be preserved during the break. Moreover, DSBs produced by Tal-effector nucleases in humans do not yield insertions that form tandem repeats, despite the fact that the breaks generate a 5′ overhang (15). Thus, this common class of mutations currently lacks a firm molecular explanation.Similar to tandem duplications, short duplications are commonly found within 100 bp of one another, but with unique intervening DNA (16). By comparing human polymorphisms with chimp sequence, Thomas et al. (16) inferred that the repeats were recent insertions. As discussed by the authors and herein, a mechanism for such duplications is even less forthcoming than for tandem duplications.In this study, we used extensive population-scale rice resequencing data to confirm that tandem duplications are also abundant natural polymorphisms in the plant kingdom. Additionally, we found that many insertions in rice, although not perfectly tandem, are from a ∼50-bp window around the insertion site. We rarely found the end homology in tandem repeats that is expected for replication slippage, although we did note a bias toward short microhomology between insertion ends and insertion site. These data led us to elaborate on the DSB model of tandem duplication, proposing that long patch base excision repair (BER) on complementary strands commonly leads to such patterns (17). Additionally, we characterized common forms of nontandem, but local, duplication.     

Template disruptions and failure of double Holliday junction dissolution during double-strand break repair in Drosophila BLM mutants

Previous biochemical studies of the BLM gene product have shown its ability in conjunction with topoisomerase IIIalpha to resolve double Holliday structures through a process called "dissolution." This process could prevent crossing over during repair of double-strand breaks. We report an analysis of the Drosophila BLM gene, DmBlm, in the repair of double-strand breaks in the premeiotic germ line of Drosophila males. With a repair reporter construct, Rr3, and other genetic tools, we show that DmBlm mutants are defective for homologous repair but show a compensating increase in single-strand annealing. Increases of 40- to 50-fold in crossing over and flanking deletions also were seen. Perhaps most significantly, the template used for homologous repair in DmBlm mutants is itself subject to deletions and complex rearrangements. These template disruptions are indicative of failure to resolve double Holliday junctions. These findings, along with the demonstration that a weak allele of topoisomerase IIIalpha has some of the same defects as DmBlm, support the dissolution model. Finally, an analysis of DmBlm mutants in conjunction with mus81 or spnA (Rad51) reveals a second function of BLM distinct from the repair of induced double-strand breaks and possibly related to maintenance of replication forks.     

Light, but not heavy alcohol drinking, stimulates paraoxonase by upregulating liver mRNA in rats and humans

Paraoxonase 1 (PON) may contribute to the cardioprotective action of high-density lipoprotein (HDL) because it inhibits low-density lipoprotein (LDL) oxidation, a prerequisite for the onset of atherosclerosis. Because light drinking and heavy drinking have diametrically opposite effects on cardioprotection, we have determined the effects of ethanol dosage on rat serum PON activity and its hepatic expression. Furthermore, we have investigated PON activity and polymorphism in human light and heavy drinkers. Our results confirm that HDL-PON inhibited LDL oxidation, destroyed oxidized LDL, and inhibited its uptake by macrophages. Light ethanol feeding caused a 20% to 25% (P <.05) increase in PON activity in both serum and liver and a 59% (P <.001) increase in the level of liver PON mRNA compared with pair-fed control rats. In contrast, heavy ethanol feeding caused a 25% (P <.05) decrease in serum and liver PON activities with a 51% (P <.01) decrease in liver PON mRNA level. Light drinkers had a 395% (P <.001) higher, whereas heavy drinkers had a 45% (P <.001) lower serum PON activity compared with nondrinkers. Significantly, the number of homozygotes versus heterozygotes with respect to high or low activity PON phenotype was similar in all the groups. Therefore, we conclude that light drinking upregulates, whereas heavy drinking downregulates PON activity and its expression, irrespective of its genetic polymorphism.