Determination of Structural Polysaccharides and Lignln in Cattail Biomass

Preliminary research in this lab suggested that cattail biomass was a possible resource for the production of cellulosic ethanol. To rigorously assess the economic potential for making ethanol from cattails. Standard Laboratory Analytical Procedures developed by the National Renewable Energy Laboratory were used to characterize the extractives, structural carbohydrates and lignin in cattail leaves. Extractives were determined by using Soxhlet extraction with water followed by ethanol. Initial trials indicated that dried cattail leaves contained 20% water extractives and 6% ethanol extractives. The dried residual solid was autoclaved with 4% sulfunc acid for one hour. The mixture filtered through a filter crucible. The recovered liquid was analyzed for acid-soluble lignins by its uv absorbance and for carbohydrates by high performance liquid chromatography. The recovered solid was analyzed for acid-insoluble lignin and ash gravimetrically

The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability

<div><p>DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the <i>Rev3l</i> gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that <i>Rev3l</i> cDNA rescues the genomic instability and DNA damage sensitivity of <i>Rev3l</i>-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of <i>REV3L</i> could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity <i>in vivo</i>, a <i>Rev3l</i> knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to <i>Rev3l</i>-null fibroblasts. We tested whether the severe <i>Rev3l</i>^-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, <i>Rev3l</i>^-/-<i>Polh</i>^-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as <i>Rev3l</i>^-/-<i>Polh</i>^+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.</p></div

Deletion of <i>Polh</i> does not ameliorate phenotypes caused by knockout of <i>Rev3l</i>.

<p>(A) Genotypes of mouse pups produced by breeding parental <i>Rev3l</i>^-/lox<i>Polh</i>^-/—mice. (B) MEFs with the indicated genotypes were stained with DAPI, and for 53BP1 and γ-H2AX by immunofluorescence as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005759#pgen.1005759.g002" target="_blank">Fig 2</a> to detect foci of DNA double-strand breaks. The quantification shows the percentage of cells with >2 53BP1 and γ-H2AX foci in <i>Rev3l</i>^-/lox<i>Polh</i>^+/+, <i>Rev3l</i>^-/lox<i>Polh</i>^-/-, <i>Rev3l</i>^-/Δ<i>Polh</i>^+/+ and <i>Rev3l</i>^-/Δ<i>Polh</i>^-/- MEFs 9 days after AdCre treatment. (C) Survival of primary MEFs as in part B, 120 hr after addition of cisplatin, as measured by ATP content. For panels B& C, Data represent mean ± SEM.</p

Expression of human <i>REV3L</i> complements <i>Rev3l</i>-deficient MEFs.

<p>(A) Top, the human <i>REV3L</i> gene was cloned into a pOZ vector for expression in mammalian cells with an N-terminal FLAG-HA epitope tag. The vector also expresses the interleukin 2 receptor (<i>IL2R</i>) gene via an internal ribosomal entry site (IRES). Below, domains in mammalian REV3L protein. Indicated here are the N-terminal domain, positively-charged domain, two REV7-binding domains, the KIAA2022 homology domain (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005759#pgen.1005759.s001" target="_blank">S1 Fig</a>), and the C-terminal Fe-S cluster for interaction with other subunits. Vertical bars in the polymerase domain represent highly conserved motifs. The location of the D2781A/D2783A active site mutations (ASM) is shown. (B) Expression of <i>REV3L</i> in MEF cell lines. A set of primers and a Taqman probe were used that recognizes both human and mouse <i>Rev3l</i>, but does not amplify knockout transcript. Functional mouse <i>Rev3l</i> is expressed in <i>Rev3l</i>^+/+ but not <i>Rev3l</i>^-/Δ cells. Wild-type or ASM recombinant <i>REV3L</i> mRNA was expressed in immortalized <i>Rev3l</i>^-/Δ MEFs at about half of the endogenous level. (C) Doubling time (in hr) of MEFs harboring empty vector (EV) <i>Rev3l</i>^+/Δ, <i>Rev3l</i>^-/Δ, and <i>Rev3l</i>^-/Δ MEFs expressing wild-type or ASM recombinant <i>REV3L</i>. (D) Survival of MEFs harboring empty vector (EV): <i>Rev3l</i>^+/Δ (maroon), <i>Rev3l</i>^-/Δ (orange); and <i>Rev3l</i>^-/Δ MEFs expressing wild-type (green) or ASM (blue) recombinant <i>REV3L</i>. ATP content was measured 48 hr after addition of cisplatin. (E) Survival of these cell lines 48 hr after ultraviolet C (UVC) radiation as measured by ATP content. Data represent mean ± SEM.</p

Knock-in mice and MEFs expressing active site mutant <i>Rev3l</i> have knockout phenotypes.

<p>(A) Diagram of the mouse <i>Rev3l</i> ASM knock-in allele, with the wild-type (WT) locus shown at the top. Green rectangles indicate <i>Rev3l</i> coding sequences and the gray line represents chromosomal sequence. In the middle diagram, FRT sites are represented by double red triangles, loxP sites by blue triangles and lox511 sites by yellow triangles. The targeted exon 27 (starred) carries D2773A and D2775A point mutations and is inserted in an inverted orientation between wild-type exons 26 and 27. Splicing donor and acceptor sites flanking wild-type and ASM exon 27 are kept intact. The knock-in was produced by a Cre-dependent genetic switch. First, the neomycin positive selection cassette (neo) was excised by breeding with C57BL/6 Flp deleter mice. A subsequent cross with Cre-expressing mice led to excision of the wild-type exon 27 and inversion of ASM mutant exon 27 into the functional orientation. In the constitutive ASM knock-in locus shown in the lower diagram, the D2773A/D2775A <i>Rev3l</i> gene is expressed under the control of the endogenous <i>Rev3l</i> promoter and wild-type <i>Rev3l</i> exon 27 is absent from the locus. Heterozygous ASM knock-in mice (<i>Rev3L</i>^+/M) were then used for breeding. (B) Example of Southern blot analysis of (left) the inducible knock-in locus (<i>neo</i>⁺) and (right) the constitutive ASM knock-in locus. Genomic DNA of the tested animals was compared with C57BL/6 wild-type genomic DNA (WT). <i>Eco</i>RV digested DNA was blotted on a nylon membrane and hybridized with the external 3’ probe with the position shown at the top of part A. Restriction fragments of 15 kb, 11.5 kb and 9.5 kb were observed for the wild-type, inducible knock-in locus (<i>neo</i>⁺) and constitutive ASM knock-in locus, respectively. Genomic DNA was further analyzed extensively and confirmed by specific PCR assays and complete DNA sequencing as described in the Materials and Methods. (C) Genotypes of mouse pups produced by breeding parental <i>Rev3l</i>^+/M mice. (D) Growth of <i>Rev3l</i>^+/Δ and <i>Rev3l</i>^M/Δ cells. These cells were produced by addition of AdCre to <i>Rev3l</i>^M/lox or <i>Rev3l</i>^+/lox MEFs, deleting the floxed allele of <i>Rev3l</i>. (E) Survival of <i>Rev3l</i>^+/lox, <i>Rev3l</i>^M/lox, <i>Rev3l</i>^+/Δ and <i>Rev3l</i>^M/Δ primary MEFs 120 hr after addition of cisplatin, as measured by ATP content. (F) The MEFs as in (E) were stained with DAPI, and for 53BP1 and γ-H2AX by immunofluorescence as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005759#pgen.1005759.g002" target="_blank">Fig 2</a> to detect foci of DNA double-strand breaks. The quantification shows the percentage of cells with >2 53BP1 and γ-H2AX foci in <i>Rev3l</i>^+/lox, <i>Rev3l</i>^M/lox, <i>Rev3l</i>^+/Δ and <i>Rev3l</i>^M/Δ primary MEFs 9 days after AdCre treatment. (*) p < 0.01. Data represent mean ± SEM.</p

Spontaneous DNA double-strand break formation is reduced in <i>Rev3l</i> complemented MEFs.

<p>(A) DAPI staining of empty vector (EV)-expressing <i>Rev3l</i>^+/Δ, <i>Rev3l</i>^-/Δ, as well as <i>Rev3l</i>^-/Δ MEFs expressing wild-type or ASM recombinant <i>REV3L</i>; arrows indicate micronuclei, with an enlarged example in the inset. (B) Merged immunofluorescence staining of the same MEFs as in (A) with DAPI (blue), 53BP1 (red) and γ-H2AX (green); foci indicate areas of DNA double-strand breaks. (C) Quantification of percent of nuclei that have associated micronuclei. (D) Quantification of cells with fewer than 3, 3 to 5, 6 to 10 or greater than 10 53BP1 foci (as measured using CellProfiler). The bars are color-coded exactly as in Part C to indicate the genotype of the MEFs (*) p < 0.01. Data represent mean ± SEM.</p

POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis

BRCA2-mutant cells are defective in homologous recombination, making them vulnerable to the inactivation of other pathways for the repair of DNA double-strand breaks (DSBs). This concept can be clinically exploited but is currently limited due to insufficient knowledge about how DSBs are repaired in the absence of BRCA2. We show that DNA polymerase θ (POLθ)-mediated end joining (TMEJ) repairs DSBs arising during the S phase in BRCA2-deficient cells only after the onset of the ensuing mitosis. This process is regulated by RAD52, whose loss causes the premature usage of TMEJ and the formation of chromosomal fusions. Purified RAD52 and BRCA2 proteins both block the DNA polymerase function of POLθ, suggesting a mechanism explaining their synthetic lethal relationships. We propose that the delay of TMEJ until mitosis ensures the conversion of originally one-ended DSBs into two-ended DSBs. Mitotic chromatin condensation might further serve to juxtapose correct break ends and limit chromosomal fusions

DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability

International audienceThe DNA polymerase zeta (Polf) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polf, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polf/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-stranded breaks, eliciting deletions in late-replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polf, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polf in preventing chromosome instability during replication of heterochromatic regions

Analysis of DNA polymerase ν function in meiotic recombination, immunoglobulin class-switching, and DNA damage tolerance

<div><p>DNA polymerase ν (pol ν), encoded by the <i>POLN</i> gene, is an A-family DNA polymerase in vertebrates and some other animal lineages. Here we report an in-depth analysis of pol ν–defective mice and human cells. <i>POLN</i> is very weakly expressed in most tissues, with the highest relative expression in testis. We constructed multiple mouse models for <i>Poln</i> disruption and detected no anatomic abnormalities, alterations in lifespan, or changed causes of mortality. Mice with inactive <i>Poln</i> are fertile and have normal testis morphology. However, pol ν–disrupted mice have a modestly reduced crossover frequency at a meiotic recombination hot spot harboring insertion/deletion polymorphisms. These polymorphisms are suggested to generate a looped-out primer and a hairpin structure during recombination, substrates on which pol ν can operate. Pol ν-defective mice had no alteration in DNA end-joining during immunoglobulin class-switching, in contrast to animals defective in the related DNA polymerase θ (pol θ). We examined the response to DNA crosslinking agents, as purified pol ν has some ability to bypass major groove peptide adducts and residues of DNA crosslink repair. Inactivation of <i>Poln</i> in mouse embryonic fibroblasts did not alter cellular sensitivity to mitomycin C, cisplatin, or aldehydes. Depletion of <i>POLN</i> from human cells with shRNA or siRNA did not change cellular sensitivity to mitomycin C or alter the frequency of mitomycin C-induced radial chromosomes. Our results suggest a function of pol ν in meiotic homologous recombination in processing specific substrates. The restricted and more recent evolutionary appearance of pol ν (in comparison to pol θ) supports such a specialized role.</p></div