RPA and POT1: Friends or foes at telomeres?

Telomere maintenance in cycling cells relies on both DNA replication and capping by the protein complex shelterin. Two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomere 1 (POT1) play critical roles in DNA replication and telomere capping, respectively. While RPA binds to ssDNA in a non-sequence-specific manner, POT1 specifically recognizes singlestranded TTAGGG telomeric repeats. Loss of POT1 leads to aberrant accumulation of RPA at telomeres and activation of the ataxia telangiectasia and Rad3-related kinase (ATR)-mediated checkpoint response, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. The requirement for both POT1 and RPA in telomere maintenance and the antagonism between the two proteins raises the important question of how they function in concert on telomeric ssDNA. Two interesting models were proposed by recent studies to explain the regulation of POT1 and RPA at telomeres. Here, we discuss how these models help unravel the coordination, and also the antagonism, between POT1 and RPA during the cell cycle.Key words: RPA, POT1, telomere, ATR, checkpointTelomeres, the natural ends of chromosomes, are composed of repetitive DNA sequences and “capped” by both specific proteins and non-coding RNAs.^1–3 One of the critical functions of telomeres is to prevent chromosomal ends from recognition by the DNA damage response machinery. Critically short or improperly capped telomeres lead to telomere dysfunction and are a major source of genomic instability.⁴ While telomeres need to be properly capped to remain stable, they also need to be duplicated during each cell division by the DNA replication machinery. The requirement of these two seemingly competing processes for telomere maintenance suggests that the cell must coordinate DNA replication and capping of telomeres to ensure faithful telomere duplication yet avoid an inappropriate DNA damage response.Telomeric DNA is unique in several ways. The bulk of each human telomere is comprised of double-stranded TTA GGG repeats. At the very end of each telomere, a stretch of single-stranded TTAGGG repeats exists as a 3′ overhang. The TTA GGG repeats in the telomeric single-stranded DNA (ssDNA) allow it to loop back and invade telomeric double-stranded DNA (dsDNA), forming a structure called the t-loop.⁵ At the base of the t-loop, the TTAGGG strand of the telomeric dsDNA is displaced by the invading single-stranded 3′ overhang to form a single-stranded D-loop. Thus, the unique DNA sequence and structures of telomeres confer the ability to bind proteins in both sequence- and structure-specific manners, providing the basis for additional regulations.In human cells, telomere capping is orchestrated by the protein complex shelterin, which contains TRF1, TRF2, RAP1, TIN2, TPP1 and POT1.³ Among these shelterin components, TRF1 and TRF2 interact with telomeric dsDNA in a sequence-specific manner, whereas POT1, in a complex with TPP1, binds to telomeric ssDNA in a sequence-specific manner.^6–8 While the human genome contains only one POT1 gene, the mouse genome contains two POT1-related genes, POT1a and POT1b.^9–11 TIN2 functions to stabilize TRF1 and TRF2 DNA binding and also tethers the POT1-TPP1 heterodimer to the rest of the shelterin complex on telomeric dsDNA.^12,13Unlike the properly capped telomeres, double-stranded DNA breaks (DSBs) with ssDNA overhangs are known to activate the ATR checkpoint kinase.^14,15 In a complex with its functional partner ATRIP, ATR is recruited to ssDNA by RPA, a non-sequence-specific ssDNA-binding protein complex.¹⁶ In addition to the ATR-ATRIP kinase complex, several other checkpoint proteins involved in ATR activation are also recruited in the presence of RPA-ssDNA.¹⁵ The structural resemblance between DSBs and telomeres and the presence of ssDNA at telomeres raise the important question as to how ATR activation is repressed at telomeres.     

Hypoxia: A novel function for VIN3

VERNALIZATION INSENSITIVE 3 (VIN3) encodes a PHD domain chromatin remodelling protein that is induced in response to cold and is required for the establishment of the vernalization response in Arabidopsis thaliana.¹ Vernalization is the acquisition of the competence to flower after exposure to prolonged low temperatures, which in Arabidopsis is associated with the epigenetic repression of the floral repressor FLOWERING LOCUS C (FLC).^2,3 During vernalization VIN3 binds to the chromatin of the FLC locus,¹ and interacts with conserved components of Polycomb-group Repressive Complex 2 (PRC2).^4,5 This complex catalyses the tri-methylation of histone H3 lysine 27 (H3K27me3),^4,6,7 a repressive chromatin mark that increases at the FLC locus as a result of vernalization.^4,7–10 In our recent paper¹¹ we found that VIN3 is also induced by hypoxic conditions, and as is the case with low temperatures, induction occurs in a quantitative manner. Our experiments indicated that VIN3 is required for the survival of Arabidopsis seedlings exposed to low oxygen conditions. We suggested that the function of VIN3 during low oxygen conditions is likely to involve the mediation of chromatin modifications at certain loci that help the survival of Arabidopsis in response to prolonged hypoxia. Here we discuss the implications of our observations and hypotheses in terms of epigenetic mechanisms controlling gene regulation in response to hypoxia.Key words: arabidopsis, VIN3, FLC, hypoxia, vernalization, chromatin remodelling, survival     

Human non-CG methylation: Are human stem cells plant-like?

Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.¹ DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.²Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.³ Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,^4,5 or transposons and repetitive sequences such as the human L1 retrotransposon⁶ in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.⁶Non-CG methylation has also been reported at origins of replication^7,8 and a region of the human myogenic gene Myf3.⁹ The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.¹⁰ The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.¹⁰ The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.¹¹ Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.¹² Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.¹³ Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.¹³ This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.¹⁴ confirmed a much earlier report by Ramsahoye et al.¹⁵ suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,¹⁵ thus global methylation patterning was assessed. Lister et al.¹⁴ extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report¹⁴ that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,¹⁴ one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.¹⁴Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,^14,15 raises the possibility that cancer stem cells¹⁶ carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing⁸ or ligation mediated PCR¹⁷ methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster¹⁸ or human breast cancer DNA¹³ treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand¹⁸ or at a symmetrical CWG site.¹³ In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.¹³In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,^14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family¹⁹ and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.¹⁵ DRM proteins however, possess a unique permuted domain structure found exclusively in plants¹⁹ and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published^20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies²⁴ or find that CG methylation seen in plants but not non-CG methylation is detected.^21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,²⁷ and is consistent with the recent report that cultured stem cells are epigenetically unstable.²⁸The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.^9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes²⁹ even though the promoters were symmetrically de novo methylated at ^mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.^9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.¹⁴ Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,²⁹ however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.^30–33 However, depletion of CWG sites is not observed in the human genome.³⁴ Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome¹⁴ where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome¹⁴ raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.¹⁴ This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.¹⁴By analogy with the well known methylated DNA binding proteins specific for CG methylation,³⁵ methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.^36–38 While Dnmt1 has been proposed to function stoichiometrically³⁹ and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,⁴⁰ a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design^41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.^14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR⁴³ the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche^44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,⁴⁶ COMPARE-MS⁴⁷) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP⁴⁸) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.¹⁴In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function.     

Timing the switch to phototrophic growth: A possible role of GUN1

In young Arabidopsis seedlings, retrograde signaling from plastids regulates the expression of photosynthesis-associated nuclear genes in response to the developmental and functional state of the chloroplasts. The chloroplast-located PPR protein GUN1 is required for signalling following disruption of plastid protein synthesis early in seedling development before full photosynthetic competence has been achieved. Recently we showed that sucrose repression and the correct temporal expression of LHCB1, encoding a light-harvesting chlorophyll protein associated with photosystem II, are perturbed in gun1 mutant seedlings.¹ Additionally, we demonstrated that in gun1 seedlings anthocyanin accumulation and the expression of the “early” anthocyanin-biosynthesis genes is perturbed. Early seedling development, predominantly at the stage of hypocotyl elongation and cotyledon expansion, is also affected in gun1 seedlings in response to sucrose, ABA and disruption of plastid protein synthesis by lincomycin. These findings indicate a central role for GUN1 in plastid, sucrose and ABA signalling in early seedling development.Key words: ABA, ABI4, anthocyanin, chloroplast, GUN1, retrograde signalling, sucroseArabidopsis seedlings develop in response to light and other environmental cues. In young seedlings, development is fuelled by mobilization of lipid reserves until chloroplast biogenesis is complete and the seedlings can make the transition to phototrophic growth. The majority of proteins with functions related to photosynthesis are encoded by the nuclear genome, and their expression is coordinated with the expression of genes in the chloroplast genome. In developing seedlings, retrograde signaling from chloroplasts to the nucleus regulates the expression of these nuclear genes and is dependent on the developmental and functional status of the chloroplast. Two classes of gun (genomes uncoupled) mutants defective in retrograde signalling have been identified in Arabidopsis: the first, which comprises gun2–gun5, involves mutations in genes encoding components of tetrapyrrole biosynthesis.^2,3 The other comprises gun1, which has mutations in a nuclear gene encoding a plastid-located pentatricopeptide repeat (PPR) protein with an SMR (small MutS-related) domain near the C-terminus.^4,5 PPR proteins are known to have roles in RNA processing⁶ and the SMR domain of GUN1 has been shown to bind DNA,⁴ but the specific functions of these domains in GUN1 are not yet established. However, GUN1 has been shown to be involved in plastid gene expression-dependent,⁷ redox,⁴ ABA1,⁴ and sucrose signaling,^1,4,8 as well as light quality and intensity sensing pathways.^9–11 In addition, GUN1 has been shown to influence anthocyanin biosynthesis, hypocotyl extension and cotyledon expansion.^1,11     

Mismatch repair outside of replication

Comment on: Rodriguez GP, et al. Proc Natl Acad Sci USA 2012; 109:6153-8.DNA mismatch repair (MMR) is important for preventing mutations due to errors occurring during replication. Mismatches that are not removed by the proofreading function of the replicative DNA polymerases are recognized by a MutS protein in bacteria or usually in eukaryotes by a MutSα or MutSβ heterodimer.¹ After recognition, downstream events involving MutL in bacteria and usually MutLα in eukaryotes result in the removal of the newly synthesized DNA strand and resynthesis of the region, avoiding a change in DNA sequence.¹ However, there are many situations in which MMR is active outside of the context of genome replication, and an important question is what determines which strand of DNA is removed in those cases once a mismatch is detected. Using a reversion assay that detects point mutations in the TRP5 gene of yeast, we recently demonstrated that mispairs that escape detection at the replication fork can be recognized later by MMR and repaired in a manner independent of the replicating strand.² In that case, MMR was found to be acting in a mutagenic manner and could restore growth to cells that were in a nondividing state.When a mismatch is detected, how does MMR determine which strand to remove? A variety of experiments have demonstrated that eukaryotic MMR is directed to remove the DNA strand containing a nick, which could occur in the newly synthesized DNA strand through a variety of processes.^1,3 Evidence suggests that an important source of those nicks is activation of the latent endonuclease activity of MutLα; MutLα interacts with PCNA in a manner that could discriminate the template and primer stands of replication and thus give proper strand discrimination to MMR.⁴ Strand discrimination also likely involves MutSα and MutSβ, as both complexes have been shown to have robust interactions with PCNA.¹ However, two MMR pathways involving MutSα have recently been observed: one with MutSα as an integral component of replication factories and the other in which MutSα presumably scans the genome for mismatches and is independent of PCNA interaction.⁵ This latter pathway of MutSα, untethered to normal replication, is of interest here.One major function of MMR outside of replication is its anti-recombination role, by which it prevents aberrant recombination between non-identical sequences.⁶ MMR also functions in meiotic recombination and is responsible for the formation of gene conversion gradients, which can best be understood as arising from directed MMR near the site of initiating double-strand breaks, and randomly directed MMR further from the site of such strand discrimination signals.⁷ Although in meiotic recombination it appears that MMR acts without strand discrimination in some cases, the result is not an increase in mutation, as the end result is a choice between two pre-existing alleles.There are at least three situations in which MMR acting outside of replication appears to play a pro-mutagenic role. One is in the expansion of triplet repeat sequences, which play a role in a number of neurodegenerative diseases.^3,4 Although MMR would be expected to prevent repeat slippage during replication, there is biochemical evidence that slippage loops formed in triplet repeat sequences in a non-replicating state could load a PCNA-MutLα complex that would nick DNA in a random manner, leading to large expansions.⁴ There is also evidence from various mouse model systems that suggest a role for MMR in promoting repeat expansions.^3,8The second situation is somatic hypermutation. It had been found in 1998 that somatic hypermutation was, contrary to expectations, dependent on the presence of active MMR,⁹ but there was no mechanistic way to explain that dependence. A combination of patch replication by a relatively inaccurate translesion DNA polymerase and MMR acting randomly in terms of DNA strand, offers the best rationale for this process.^2,3The third situation, which also has the broadest sweep, is that of cancer. If several different pathways must be altered by mutation in order for a tumor to form, how could that happen in cells that either divide very slowly, or are in a nondividing state? Two years after the initial discovery of the linkage between MMR defects and cancer, MacPhee hypothesized that MMR acting in a “randomly templated” mode could be responsible for the formation of mutations in nondividing cells, which could reenter a growth phase as a result of those mutations;¹⁰ that corresponds to the observation we recently made in yeast, illustrated in Figure 1.² This is precisely the type of activity envisioned by MacPhee. What is not yet known is the signal giving strand discrimination to MMR; it could possibly be a random loading of MutLα,⁴ or it could be the presence of a random nick in the DNA strand close enough to the mismatch to be used for strand discrimination.Open in a separate windowFigure 1. Mutagenic MMR in nondividing cells. Cells were electroporated with 8-oxoGTP and then plated on Trp medium. The 8-oxoGTP was incorporated throughout the genome, including in some cells at the position indicated above which must revert via a TA→GC transversion in order for the cell to become Trp+. Once plated, the Trp- cells were unable to undergo even one round of replication, although they remained viable for over two weeks. In the absence of MMR, very few revertants appeared. However, in the presence of MMR, many revertants arose within the 3 d expected for cells that would have begun growth immediately after plating, but an even larger number of revertant colonies arose later on the plates up to a period of a week later. Thus only cells with active MMR were able to regain growth from a nondividing state due to the mutation created by MMR activity.The importance of MMR in preventing mutations during replication is unquestioned. However, the multiple activities of MMR outside of replication tend to be less appreciated. When MMR recognizes mismatches in DNA outside of the context of replication, the issue of what gives strand discrimination to MMR becomes critical. If the signal is randomly generated, MMR activity can be mutagenic, as we have found.²     

A role for PCNA2 in translesion synthesis by Arabidopsis thaliana DNA polymerase η

Eukaryotic DNA polymerase η (Polη) confers ultraviolet (UV) resistance by catalyzing translesion synthesis (TLS) past UV photoproducts. Polη has been studied extensively in budding yeast and mammalian cells, where its interaction with monoubiquitylated proliferating cell nuclear antigen (PCNA) is necessary for its biological activity. Recently, in collaboration with other investigators, our laboratory demonstrated that Arabidopsis thaliana Polη is required for UV resistance in plants. Furthermore, the purified enzyme can perform TLS opposite a cyclobutane pyrimidine dimer and interacts with PCNA. Intriguingly, the biological activity of Polη in a heterologous yeast assay depends on co-expression with Arabidopsis PCNA2 and Polη sequences implicated in binding PCNA or ubiquitin. We suggest that interaction of Arabidopsis Polη with ubiquitylated PCNA2 is required for TLS past UV photoproducts by Polη.Key words: polymerase η, proliferating cell nuclear antigen, translesion synthesis, ubiquitin, Arabidopsis thaliana, ultraviolet radiationUltraviolet (UV)-induced pyrimidine dimers can block the progression of DNA replication forks potentially disrupting the replication machinery and resulting in cell death. For this reason, cells have evolved non-essential, low fidelity DNA polymerases (Pols) capable of copying damaged templates,^1,2 a process termed translesion DNA synthesis (TLS). In budding yeast, TLS past UV photoproducts is catalyzed by Polη and Polζ (composed of the Rev3 catalytic and Rev7 accessory subunits), but also involves the Rev1 protein in an as yet undetermined role linked to Polζ.^1,3,4 Yeast and human Polη replicates cyclobutane pyrimidine dimers (CPDs), in particular thymine-thymine (TT) CPDs, in a relatively error-free manner whereas Polζ is essential for UV mutagenesis implicating it in error-prone TLS.^1,4,5Both UV resistance due to TLS and the polymerases responsible have been well-studied in yeast and mammalian cells over the past decade. Only more recently has evidence emerged that TLS may also contribute to UV resistance in plants. Arabidopsis thaliana POLH, REV1, REV3 and REV7 encode homologs of Polη, Rev1, Rev3 and Rev7, respectively.^6–10 T-DNA insertions in POLH, REV1 or REV3 sensitise root growth to acute UV doses,^6–8,10 and these mutations, as well as inactivation of REV7, increase the sensitivity of whole plants to longer term UV treatment.^6,8 Interestingly, polh rev3 double mutants show an additive increase in UV sensitivity over that observed for polh and rev3 single mutants,^6,10 potentially pointing to differences in the UV photoproducts bypassed by the two polymerases. That the enhanced UV sensitivity of the mutants may reflect a TLS deficiency is suggested by the finding that purified Arabidopsis Polη catalyzes primer extension and TLS past a TT CPD in vitro.⁶For TLS to occur, Polη must gain access to the replication machinery arrested at a UV photoproduct. It does so in yeast and mammalian cells by interacting with proliferating cell nuclear antigen (PCNA), the eukaryotic sliding clamp required for processive DNA replication.^1,3,11, DNA damage or stalling of the replicative polymerase triggers monoubiquitylation of PCNA at lysine 164 by a complex of the E2 ubiquitin conjugase Rad6 and the E3 ubiquitin ligase Rad18.^1,3,11,12 This modification increases the affinity of Polη for PCNA, with which it interacts via a single PCNA interacting peptide (PIP) box and a single ubiquitin-binding zinc finger (UBZ) domain.^1,3In contrast to its yeast and mammalian counterparts, Polη from Arabidopsis and Oryza sativa (rice) has two PIP boxes and lacks a UBZ.^6,9,10 Instead the two polymerases each possess two ubiquitin-binding motifs (UBMs) similar to those present in the Arabidopsis Rev1 protein and a vertebrate TLS polymerase, Pol., for which there is no homolog in Arabidopsis.^6,13 Considerable differences in the sequences flanking the UBMs in Polη and Rev1 argue that Polη did not acquire its UBMs from Rev1, and so, although perhaps unique to plant Polη, their origin remains a mystery.The presence of PCNA- and ubiquitin-binding sequences in plant Polη hint that it may operate in TLS in a manner similar to that for Polη from yeast or mammalian cells. Indeed, three lines of evidence⁶ lead us to suggest that the Polη PIP boxes and UBMs likely function in binding ubiquitylated PCNA and this interaction is probably required for TLS past UV photoproducts by Arabidopsis Polη. First, Arabidopsis Polη interacts physically and in yeast two-hybrid assays with Arabidopsis PCNA1 and PCNA2. Second, expression in yeast of Arabidopsis cDNAs encoding Polη and PCNA2, but not PCNA1, fully complements the UV sensitivity conferred by elimination of yeast Polη. In vitro mutagenesis suggests the inability of Polη plus PCNA1 to restore UV resistance is due to a lysine at position 201 in PCNA1 but not PCNA2. In the three-dimensional structure of PCNA, amino acid 201 lies adjacent to lysine-164, the residue that is ubiquitylated in yeast and human PCNA. Thus, one possibility is that lysine-201 in PCNA1 prevents complementation of UV sensitivity by inhibiting ubiquitylation of lysine-164. Third, altering presumed critical residues in either of the two PIP boxes or UBM2 in Arabidopsis Polη also prevents restoration of UV resistance in Polη-deficient yeast cells.Several important parts of the puzzle remain to be solved. In particular, the ubiquitylation of plant PCNA has yet to be demonstrated, and the identity of the proteins that might monoubiquitylate plant PCNA is uncertain. Although Arabidopsis Rad6 homologs can ubiquitylate target proteins in vitro, there is no evidence that Arabidopsis PCNA1 or PCNA2 is a substrate, and Arabidopsis lacks a Rad18 homolog.^14,15 Finally, if PCNA is ubiquitylated in planta, does this occur at lysine-164 in response to DNA damage or replication fork stalling, is the interaction of Polη with PCNA stimulated by this modification, and is an enhanced interaction mediated by the Polη UBMs?     

Complete Genome Sequence of Croceibacter atlanticus HTCC2559T

Here we announce the complete genome sequence of Croceibacter atlanticus HTCC2559^T, which was isolated by high-throughput dilution-to-extinction culturing from the Bermuda Atlantic Time Series station in the Western Sargasso Sea. Strain HTCC2559^T contained genes for carotenoid biosynthesis, flavonoid biosynthesis, and several macromolecule-degrading enzymes. The genome confirmed physiological observations of cultivated Croceibacter atlanticus strain HTCC2559^T, which identified it as an obligate chemoheterotroph.The phylum Bacteroidetes comprises 6 to ∼30% of total bacterial communities in the ocean by fluorescence in situ hybridization (8-10). Most marine Bacteroidetes are in the family Flavobacteriaceae, most of which are aerobic respiratory heterotrophs that form a well-defined clade by 16S rRNA phylogenetic analyses (4). The members of this family are well known for degrading macromolecules, including chitin, DNA, cellulose, starch, and pectin (17), suggesting their environmental roles as detritus decomposers in the ocean (6). Marine Polaribacter and Dokdonia species in the Flavobacteriaceae have also shown to have photoheterotrophic metabolism mediated by proteorhodopsins (11, 12).Several strains of the family Flavobacteriaceae were isolated from the Sargasso Sea and Oregon coast, using high-throughput culturing approaches (7). Croceibacter atlanticus HTCC2559^T was cultivated from seawater collected at a depth of 250 m from the Sargasso Sea and was identified as a new genus in the family Flavobacteriaceae based on its 16S rRNA gene sequence similarities (6). Strain HTCC2559^T met the minimal standards for genera of the family Flavobacteriaceae (3) on the basis of phenotypic characteristics (6).Here we report the complete genome sequence of Croceibacter atlanticus HTCC2559^T. The genome sequencing was initiated by the J. Craig Venter Institute as a part of the Moore Foundation Microbial Genome Sequencing Project and completed in the current announcement. Gaps among contigs were closed by Genotech Co., Ltd. (Daejeon, Korea), using direct sequencing of combinatorial PCR products (16). The HTCC2559^T genome was analyzed with a genome annotation system based on GenDB (14) at Oregon State University and with the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (15, 16).The HTCC2559^T genome is 2,952,962 bp long, with 33.9 mol% G+C content, and there was no evidence of plasmids. The number of protein-coding genes was 2,715; there were two copies of the 16S-23S-5S rRNA operon and 36 tRNA genes. The HTCC2559^T genome contained genes for a complete tricarboxylic acid cycle, glycolysis, and a pentose phosphate pathway. The genome also contained sets of genes for metabolic enzymes involved in carotenoid biosynthesis and also a serine/glycine hydroxymethyltransferase, which is often associated with the assimilatory serine cycle (13). The potential for HTCC2559^T to use bacterial type III polyketide synthase (PKS) needs to be confirmed because this organism had a naringenin-chalcone synthase (CHS) or chalcone synthase (EC 2.3.1.74), a key enzyme in flavonoid biosynthesis. CHS initiates the addition of three molecules of malonyl coenzyme A (malonyl-CoA) to a starter CoA ester (e.g., 4-coumaroyl-CoA) (1) and takes part in a few bacterial type III polyketide synthase systems (1, 2, 5, 18).The complete genome sequence confirmed that strain HTCC2559^T is an obligate chemoheterotroph because no genes for phototrophy were found. As expected from physiological characteristics (6), the HTCC2559^T genome contained a set of genes coding for enzymes required to degrade high-molecular-weight compounds, including peptidases, metallo-/serine proteases, pectinase, alginate lyases, and α-amylase.     

Helicase-inactivating mutations as a basis for dominant negative phenotypes

There is ample evidence from studies of both unicellular and multicellular organisms that helicase-inactivating mutations lead to cellular dysfunction and disease phenotypes. In this review, we will discuss the mechanisms underlying the basis for abnormal phenotypes linked to mutations in genes encoding DNA helicases. Recent evidence demonstrates that a clinically relevant patient missense mutation in Fanconi Anemia Complementation Group J exerts detrimental effects on the biochemical activities of the FANC J helicase, and these molecular defects are responsible for aberrant genomic stability and a poor DNA damage response. The ability of FANC J to use the energy from AT P hydrolysis to produce the force required to unwind duplex or G-quadruplex DNA structures or destabilize protein bound to DNA is required for its DNA repair functions in vivo. Strikingly, helicase-inactivating mutations can exert a spectrum of dominant negative phenotypes, indicating that expression of the mutant helicase protein potentially interferes with normal DNA metabolism and has an effect on basic cellular processes such as DNA replication, the DNA damage response and protein trafficking. This review emphasizes that future studies of clinically relevant mutations in helicase genes will be important to understand the molecular pathologies of the associated diseases and their impact on heterozygote carriers.Key words: helicase, dominant negative, dominant lethal, DNA repair, replication, genomic stability, human disease, Fanconi anemia, FANCJ, G-quadruplexClassic genetic complementation studies in E. coli provided some of the first biological evidence that the catalytic DNA unwinding activity performed by helicases is critically important in various aspects of cellular DNA metabolism including replication, DNA repair and recombination.^1,2 Appreciation that the conserved amino acid motifs found in a variety of cellular DNA helicases compose protein domains important for ATP binding and hydrolysis, DNA binding and coupling of ATP hydrolysis to separation of complementary strands in a structured nucleic acid molecule led to new insights for how helicases function mechanistically (reviewed in ref. ^3–5). Advances in understanding the roles of helicases in DNA repair really provided a foundation for future progress in the field. The genetics in bacterial systems gradually led to similar complementation experiments in the unicellular eukaryotic model organisms Sachromyces cerevisiae and Sachromyces pombe.^6–10 In a number of cases, the underlying mechanisms whereby helicases operate to preserve genomic stability were found to be generally conserved between prokaryotes and eukaryotes. Ultimately, genetic analyses of helicase mutants in higher order eukaryotes (e.g., C. elegans,^11–13 Drosophila,¹⁴ Xenopus^15,16) have begun to pave the way for studies of mammalian DNA helicases in cell-based models and mice. As human helicase genetic disorders are continuously being discovered,^17–24 the interest and pace of investigation to understand how helicases preserve the genome continues to accelerate (reviewed in refs. ^25–31).In this review, we will focus on discussing some landmark studies that describe how helicase-inactivating mutations disrupt normal cellular DNA metabolism, with a particular emphasis on dominant negative phenotypes exerted by mutations in helicase genes. This topic deserves special attention since recent evidence from our lab and others suggests that naturally occurring mutations in helicase genes exert deleterious effects even in a wild-type background which provides insight to the molecular-genetic roles of various helicases to maintain genomic stability. We will begin by discussing genetic and biochemical studies of bacterial DNA helicases which provide an excellent foundation for this line of inquiry, and then focus on eukaryotic helicases with a particular emphasis placed on human DNA helicases, mutated in chromosomal instability and DNA repair disorders.     

Neutralization of Clostridium difficile toxin A using antibody combinations

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.Key words: Clostridium difficile, toxin neutralization, therapeutic antibody, cell wall binding domains, repeat proteins, CROPs, mAb combinationThe most common cause of nosocomial antibiotic-associated diarrhea is the gram-positive, spore-forming anaerobic bacillus Clostridium difficile (C. difficile). Infection can be asymptomatic or lead to acute diarrhea, colitis, and in severe instances, pseudomembranous colitis and toxic megacolon.^1,2The pathological effects of C. difficile have long been linked to two secreted toxins, A and B.^3,4 Some strains, particularly the virulent and antibiotic-resistant strain 027 with toxinotype III, also produce a binary toxin whose significance in the pathogenicity and severity of disease is still unclear.⁵ Early studies including in vitro cell-killing assays and ex vivo models indicated that toxin A is more toxigenic than toxin B; however, recent gene manipulation studies and the emergence of virulent C. difficile strains that do not express significant levels of toxin A (termed “A⁻ B⁺”) suggest a critical role for toxin B in pathogenicity.^6,7Toxins A and B are large multidomain proteins with high homology to one another. The N-terminal region of both toxins enzymatically glucosylates small GTP binding proteins including Rho, Rac and CDC42,^8,9 leading to altered actin expression and the disruption of cytoskeletal integrity.^9,10 The C-terminal region of both toxins is composed of 20 to 30 residue repeats known as the clostridial repetitive oligopeptides (CROPs) or cell wall binding (CWB) domains due to their homology to the repeats of Streptococcus pneumoniae LytA,^11–14 and is responsible for cell surface recognition and endocytosis.^12,15–17C. difficile-associated diarrhea is often, but not always, induced by antibiotic clearance of the normal intestinal flora followed by mucosal C. difficile colonization resulting from preexisting antibiotic resistant C. difficile or concomitant exposure to C. difficile spores, particularly in hospitals. Treatments for C. difficile include administration of metronidazole or vancomycin.^2,18 These agents are effective; however, approximately 20% of patients relapse. Resistance of C. difficile to these antibiotics is also an emerging issue^19,20 and various non-antibiotic treatments are under investigation.^20–25Because hospital patients who contract C. difficile and remain asymptomatic have generally mounted strong antibody responses to the toxins,^26,27 active or passive immunization approaches are considered hopeful avenues of treatment for the disease. Toxins A and B have been the primary targets for immunization approaches.^20,28–33 Polyclonal antibodies against toxins A and B, particularly those that recognize the CWB domains, have been shown to effectively neutralize the toxins and inhibit morbidity in rodent infection models.³¹ Monoclonal antibodies (mAbs) against the CWB domains of the toxins have also demonstrated neutralizing capabilities; however, their activity in cell-based assays is significantly weaker than that observed for polyclonal antibody mixtures.^33–36We investigated the possibility of creating a cocktail of two or more neutralizing mAbs that target the CWB domain of toxin A with the goal of synthetically re-creating the superior neutralization properties of polyclonal antibody mixtures. Using the entire CWB domain of toxin A, antibodies were raised in rodents and screened for their ability to neutralize toxin A in a cell-based assay. Two mAbs, 3358 and 3359, that (1) both independently demonstrated marginal neutralization behavior and (2) did not cross-block one another from binding toxin A were identified. We report here that 3358 and 3359 use differing mechanisms to modify CWB-domain association with CHO cell surfaces and combine favorably to reduce toxin A-mediated cell lysis.     

The Role of the DNA Damage Response Kinase Ataxia Telangiectasia Mutated in Neuroprotection

It has been estimated that a human cell is confronted with 1 million DNA lesions every day, one fifth of which may originate from the activity of Reactive Oxygen Species (ROS) alone [1,2]. Terminally differentiated neurons are highly active cells with, if any, very restricted regeneration potential [3]. In addition, genome integrity and maintenance during neuronal development is crucial for the organism. Therefore, highly accurate and robust mechanisms for DNA repair are vital for neuronal cells. This requirement is emphasized by the long list of human diseases with neurodegenerative phenotypes, which are either caused by or associated with impaired function of proteins involved in the cellular response to genotoxic stress [4-8]. Ataxia Telangiectasia Mutated (ATM), one of the major kinases of the DNA Damage Response (DDR), is a node that links DDR, neuronal development, and neurodegeneration [2,9-12]. In humans, inactivating mutations of ATM lead to Ataxia-Telangiectasia (A-T) disease [11,13], which is characterized by severe cerebellar neurodegeneration, indicating an important protective function of ATM in the nervous system [14]. Despite the large number of studies on the molecular cause of A-T, the neuroprotective role of ATM is not well established and is contradictory to its general proapoptotic function. This review discusses the putative functions of ATM in neuronal cells and how they might contribute to neuroprotection.     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴