@article{lee_pascuzzi_settlage_shultz_tanurdzic_rabinowicz_menges_zheng_main_murray_et al._2010, title={Arabidopsis thaliana Chromosome 4 Replicates in Two Phases That Correlate with Chromatin State}, volume={6}, ISSN={1553-7404}, url={http://dx.doi.org/10.1371/journal.pgen.1000982}, DOI={10.1371/journal.pgen.1000982}, abstractNote={DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins.}, number={6}, journal={PLoS Genetics}, publisher={Public Library of Science (PLoS)}, author={Lee, Tae-Jin and Pascuzzi, Pete E. and Settlage, Sharon B. and Shultz, Randall W. and Tanurdzic, Milos and Rabinowicz, Pablo D. and Menges, Margit and Zheng, Ping and Main, Dorrie and Murray, James A. H. and et al.}, editor={Copenhaver, Gregory P.Editor}, year={2010}, month={Jun}, pages={e1000982} } @article{ascencio-ibanez_settlage_2007, title={DNA abrasion onto plants is an effective method for geminivirus infection and virus-induced gene silencing}, volume={142}, ISSN={["1879-0984"]}, DOI={10.1016/j.jviromet.2007.01.031}, abstractNote={Geminiviruses belong to a rapidly growing group of plant pathogens that contribute to crop losses in tropical and subtropical areas of the world. Geminivirus infection is a model for plant DNA replication and virus/host interactions. Geminiviruses are also used as vectors to induce silencing of endogenous genes in several plant species. A method was analyzed for inoculating geminiviruses using plasmid DNA rubbed onto leaves in the presence of an abrasive (DNA abrasion). Although the use of DNA abrasion to inoculate geminiviruses has been described previously, the technique has fallen out of favor and has not been systematically optimized. However, consistent efficiencies of 100% infection rates can be achieved by DNA abrasion. The symptoms of Tomato Golden Mosaic Virus or Cabbage Leaf Curl Virus infection on Nicotiana benthamiana were similar in timing and appearance to the symptoms observed in plants inoculated using Agrobacterium as the delivery method. More importantly, silencing of an endogenous gene was highly efficient when a geminivirus silencing vector was inoculated by the DNA abrasion method. Other plant species successfully inoculated with geminiviruses by DNA abrasion were Nicotiana tabacum, Capsicum annuum and Nicandra physalodes. Unfortunately, Arabidopsis thaliana could not be infected with Cabbage Leaf Curl Virus using leaf abrasion, demonstrating limitation of the method. However, leaf abrasion to inoculate geminiviruses is an easy and inexpensive method that should be considered as an accessible technique to the growing number of researchers using geminiviruses.}, number={1-2}, journal={JOURNAL OF VIROLOGICAL METHODS}, author={Ascencio-Ibanez, Jose Trinidad and Settlage, Sharon B.}, year={2007}, month={Jun}, pages={198–203} } @article{shultz_settlage_hanley-bowdoin_thompson_2005, title={A trichloroacetic acid-acetone method greatly reduces infrared autofluorescence of protein extracts from plant tissue}, volume={23}, ISSN={["0735-9640"]}, DOI={10.1007/BF02788888}, number={4}, journal={PLANT MOLECULAR BIOLOGY REPORTER}, author={Shultz, RW and Settlage, SB and Hanley-Bowdoin, L and Thompson, WF}, year={2005}, month={Dec}, pages={405–409} } @article{settlage_see_hanley-bowdoin_2005, title={Geminivirus C3 protein: Replication enhancement and protein interactions}, volume={79}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.79.15.9885-9895.2005}, abstractNote={ABSTRACT Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus , we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants. }, number={15}, journal={JOURNAL OF VIROLOGY}, author={Settlage, SB and See, RG and Hanley-Bowdoin, L}, year={2005}, month={Aug}, pages={9885–9895} } @article{arguello-astorga_lopez-ochoa_kong_orozco_settlage_hanley-bowdoin_2004, title={A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein}, volume={78}, ISSN={["1098-5514"]}, DOI={10.1128/JVI.78.9.4817-4826.2004}, abstractNote={ABSTRACT The geminivirus replication factor AL1 interacts with the plant retinoblastoma-related protein (pRBR) to modulate host gene expression. The AL1 protein of tomato golden mosaic virus (TGMV) binds to pRBR through an 80-amino-acid region that contains two highly predicted α-helices designated 3 and 4. Earlier studies suggested that the helix 4 motif, whose amino acid sequence is strongly conserved across geminivirus replication proteins, plays a role in pRBR binding. We generated a series of alanine substitutions across helix 4 of TGMV AL1 and examined their impact on pRBR binding using yeast two-hybrid assays. These experiments showed that several helix 4 residues are essential for efficient pRBR binding, with a critical residue being a leucine at position 148 in the middle of the motif. Various amino acid substitutions at leucine-148 indicated that both structural and side chain components contribute to pRBR binding. The replication proteins of the geminiviruses tomato yellow leaf curl virus and cabbage leaf curl virus (CaLCuV) also bound to pRBR in yeast dihybrid assays. Mutation of the leucine residue in helix 4 of CaLCuV AL1 reduced binding. Together, these results suggest that helix 4 and the conserved leucine residue are part of a pRBR-binding interface in begomovirus replication proteins.}, number={9}, journal={JOURNAL OF VIROLOGY}, author={Arguello-Astorga, G and Lopez-Ochoa, L and Kong, LJ and Orozco, BM and Settlage, SB and Hanley-Bowdoin, L}, year={2004}, month={May}, pages={4817–4826} } @misc{hanley-bowdoin_settlage_2004, title={Geminivirus resistant transgenic plants expressing a mutant geminivirus AL3/C3 coding sequence}, volume={6,747,188}, number={2004 June 8}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Hanley-Bowdoin, L. and Settlage, S.}, year={2004} } @misc{hanley-bowdoin_settlage_robertson_2004, title={Reprogramming plant gene expression: a prerequisite to geminivirus DNA replication}, volume={5}, ISSN={["1364-3703"]}, DOI={10.1111/J.1364-3703.2004.00214.X}, abstractNote={SUMMARYGeminiviruses constitute a large family of plant‐infecting viruses with small, single‐stranded DNA genomes that replicate through double‐stranded intermediates. Because of their limited coding capacity, geminiviruses supply only the factors required to initiate their replication and use plant nuclear DNA polymerases to amplify their genomes. Many geminiviruses replicate in differentiated cells that no longer contain detectable levels of host DNA polymerases and associated factors. To overcome this barrier, geminiviruses induce the accumulation of DNA replication machinery in mature plant cells by reprogramming host gene expression. The mammalian DNA tumour viruses activate host genes required for DNA replication by binding to the retinoblastoma protein, a negative regulator of cell cycle progression, and relieving repression through the E2F family of transcription factors. In this review, we discuss recent experiments showing that geminiviruses also modulate components of the retinoblastoma/E2F transcription regulatory network to induce quiescent plant cells to re‐enter the cell cycle and regain the capacity to support high levels of DNA replication. Regulation of the cell division cycle and its integration with developmental pathways is complex, with many factors, including hormones, sucrose and environmental signals, controlling re‐entry into the plant cell cycle. Geminivirus interactions with these regulatory networks are likely to determine if and where they can replicate their genomes in different plant tissues and hosts.}, number={2}, journal={MOLECULAR PLANT PATHOLOGY}, author={Hanley-Bowdoin, L and Settlage, SB and Robertson, D}, year={2004}, month={Mar}, pages={149–156} } @article{egelkrout_mariconti_settlage_cella_robertson_hanley-bowdoin_2002, title={Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development}, volume={14}, ISSN={["1040-4651"]}, DOI={10.1105/tpc.006403}, abstractNote={E2F transcription factors regulate genes expressed at the G1/S boundary of the cell division cycle in higher eukaryotes. Although animal E2F proteins and their target promoters have been studied extensively, little is known about how these factors regulate plant promoters. An earlier study identified two E2F consensus binding sites in the promoter of a Nicotiana benthamiana gene encoding proliferating cell nuclear antigen (PCNA) and showed that the proximal element (E2F2) is required for the full repression of PCNA expression in mature leaves. In this study, we examined the distal element (E2F1) and how it interacts with the E2F2 site to regulate the PCNA promoter. Gel shift assays using plant nuclear extracts or purified Arabidopsis E2F and DP proteins showed that different complexes bind to the two E2F sites. Mutation of the E2F1 site or both sites differentially altered PCNA promoter function in transgenic plants. As reported previously for the E2F2 mutation, the E2F1 and E2F1+2 mutations partially relieved the repression of the PCNA promoter in mature leaves. In young tissues, the E2F1 mutation resulted in a threefold reduction in PCNA promoter activity, whereas the E2F1+2 mutation had no detectable effect. The activity of E2F1+2 mutants was indistinguishable from that of E2F2 mutants. These results demonstrate that both E2F elements contribute to the repression of the PCNA promoter in mature leaves, whereas the E2F1 site counters the repression activity of the E2F2 element in young leaves.}, number={12}, journal={PLANT CELL}, author={Egelkrout, EM and Mariconti, L and Settlage, SB and Cella, R and Robertson, D and Hanley-Bowdoin, L}, year={2002}, month={Dec}, pages={3225–3236} } @article{settlage_miller_gruissem_hanley-bowdoin_2001, title={Dual interaction of a geminivirus replication accessory factor with a viral replication protein and a plant cell cycle regulator}, volume={279}, ISSN={["0042-6822"]}, DOI={10.1006/viro.2000.0719}, abstractNote={Geminiviruses replicate their small, single-stranded DNA genomes through double-stranded DNA intermediates in plant nuclei using host replication machinery. Like most dicot-infecting geminiviruses, tomato golden mosaic virus encodes a protein, AL3 or C3, that greatly enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that AL3 forms oligomers and interacts with the viral replication initiator AL1. Experiments reported here established that AL3 also interacts with a plant homolog of the mammalian tumor suppressor protein, retinoblastoma (pRb). Analysis of truncated AL3 proteins indicated that pRb and AL1 bind to similar regions of AL3, whereas AL3 oligomerization is dependent on a different region of the protein. Analysis of truncated AL1 proteins located the AL3-binding domain between AL1 amino acids 101 and 180 to a region that also includes the AL1 oligomerization domain and the catalytic site for initiation of viral DNA replication. Interestingly, the AL3-binding domain was fully contiguous with the domain that mediates AL1/pRb interactions. The potential significance of AL3/pRb binding and the coincidence of the domains responsible for AL3, AL1, and pRb interactions are discussed.}, number={2}, journal={VIROLOGY}, author={Settlage, SB and Miller, AB and Gruissem, W and Hanley-Bowdoin, L}, year={2001}, month={Jan}, pages={570–576} } @article{hanley-bowdoin_settlage_orozco_nagar_robertson_2000, title={Geminiviruses - Models for plant DNA replication, transcription and cell cycle regulation ([correction to] vol 35, pg 105, 2000)}, volume={35}, number={4}, journal={Critical Reviews in Biochemistry and Molecular Biology}, author={Hanley-Bowdoin, L. and Settlage, S. B. and Orozco, B. M. and Nagar, S. and Robertson, D.}, year={2000}, pages={U4} } @article{hanley-bowdoin_settlage_orozco_nagar_robertson_2000, title={Geminiviruses: Models for plant DNA replication, transcription, and cell cycle regulation}, volume={35}, number={2}, journal={Critical Reviews in Biochemistry and Molecular Biology}, author={Hanley-Bowdoin, L. and Settlage, S. B. and Orozco, B. M. and Nagar, S. and Robertson, D.}, year={2000}, pages={105–140} } @article{hanley-bowdoin_settlage_orozco_nagar_robertson_1999, title={Geminiviruses: Models for plant DNA replication, transcription, and cell cycle regulation {review}}, volume={18}, DOI={10.1080/07352689991309162}, abstractNote={Geminiviruses have small, single-stranded DNA genomes that replicate through double-stranded intermediates in the nuclei of infected plant cells. Viral double-stranded DNA also assembles into minichromosomes and is transcribed in infected cells. Geminiviruses encode only a few proteins for their replication and transcription and rely on host enzymes for these processes. However, most plant cells, which have exited the cell cycle and undergone differentiation, do not contain the replicative enzymes necessary for viral DNA synthesis. To overcome this barrier, geminiviruses induce the accumulation of DNA replication machinery in mature plant cells, most likely by modifying cell cycle and transcriptional controls. In animals, several DNA viruses depend on host replication and transcription machinery and can alter their hosts to create an environment that facilitates efficient viral replication. Analysis of these viruses and their proteins has contributed significantly to our understanding of DNA replication, transcription, and cell cycle regulation in mammalian cells. Geminiviruses have the same potential for plant systems. Plants offer many advantages for these types of studies, including ease of transformation, well-defined cell populations and developmental programs, and greater tolerance of cell cycle perturbation and polyploidy. Our knowledge of the molecular and cellular events that mediate geminivirus infection has increased significantly during recent years. The goal of this review is to summarize recent research addressing geminivirus DNA replication and its integration with transcriptional and cell cycle regulatory processes.}, number={1}, journal={Critical Reviews in Plant Sciences}, author={Hanley-Bowdoin, L. and Settlage, S. B. and Orozco, B. M. and Nagar, S. and Robertson, D.}, year={1999}, pages={71–106} } @article{orozco_gladfelter_settlage_eagle_gentry_hanley-bowdoin_1998, title={Multiple cis elements contribute to geminivirus origin function}, volume={242}, ISSN={["0042-6822"]}, DOI={10.1006/viro.1997.9013}, abstractNote={The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules which are dissimilar in sequence except for a highly conserved 200-bp common region that includes the origin for rolling circle replication. To better characterize the plus-strand origin, we analyzed the capacities of various TGMV common region sequences to support episomal replication in tobacco protoplasts when the viral replication proteins AL1 and AL3 were supplied in trans. These experiments demonstrated that the minimal origin is located in 89-bp common region fragment that includes the known AL1 binding motif and a hairpin structure containing the DNA cleavage site. Analyses of mutant origin sequences identified two additional cis elements--one that is required for origin activity and a second that greatly enhances replication. In contrast, a conserved partial copy of the AL1 binding site did not contribute to origin function. Mutational analysis of the functional AL1 binding site showed that both spacing and sequence of this motif are important for replication in vivo and AL1/DNA binding in vitro. Spacing changes between the AL1 binding site and hairpin also negatively impacted TGMV origin function in a position-dependent manner. Together, these results demonstrated that the organization of TGMV plus-strand origin is complex, involving multiple cis elements that are likely to interact with each other during initiation of replication.}, number={2}, journal={VIROLOGY}, author={Orozco, BM and Gladfelter, HJ and Settlage, SB and Eagle, PA and Gentry, RN and Hanley-Bowdoin, L}, year={1998}, month={Mar}, pages={346–356} } @article{settlage_kwanyuen_wilson_1998, title={Relation between diacylglycerol acyltransferase activity and oil concentration in soybean}, volume={75}, DOI={10.1007/s11746-998-0225-2}, abstractNote={AbstractDiacylglycerol acyltransferase (EC 2.3.1.20; DGAT) catalyzes synthesis of triacylglycerol from acyl‐CoA and diacylglycerol. Activity of this enzyme and developmental changes in oil accumulation were estimated at various stages of seed growth in soybean germplasm with phenotypic differences in oil content. Oil deposition in seed of these genotypes followed a sigmoid pattern that was modeled to predict incremental rates of oil accumulation during seed development. A strong positive correlation was found between the estimated peak rate of oil deposition (near the mid‐term of seed development) and oil concentration in mature seed. At saturating substrate levels, DGAT activity measured near the peak rate of oil deposition also was correlated positively with oil phenotype. In the latter stages of seed development, a positive correlation between estimates of enzyme activity at or below the apparent Km for diolein and comparable oil accumulation rates was attributed to reduced synthesis of substrates and/or potential change in affinity for substrate as suggested by an increase in apparent Km for diolein in older seed. These data indicated that DGAT activity may be a rate‐limiting step in triacylglycerol synthesis. However, it is difficult to accept the idea of a single rate‐limiting step at the end of a complex metabolic pathway. Because oil is a quantitatively inherited trait, several genes determine genotypic differences in oil content among soybeans. Hence, DGAT activity may be an indicator of coordinated genetic expression of gene‐products in the entire glycerolipid synthetic pathway for a given genotype. In any case, results of this investigation demonstrated that genotypic differences in DGAT activity contributed to expression of genetic variation in oil content among soybean gemplasm.}, number={7}, journal={Journal of the American Oil Chemists Society}, author={Settlage, S. B. and Kwanyuen, P. and Wilson, R. F.}, year={1998}, pages={775–781} } @article{orozco_miller_settlage_hanley-bowdoin_1997, title={Functional domains of a geminivirus replication protein}, volume={272}, DOI={10.1074/jbc.272.15.9840}, abstractNote={Tomato golden mosaic virus, a member of the geminivirus family, has a single-stranded DNA genome that is replicated and transcribed in infected plant cells through the concerted action of viral and host factors. One viral protein, AL1, contributes to both processes by binding to a directly repeated, double-stranded DNA sequence located in the overlapping (+) strand origin of replication and AL1 promoter. The AL1 protein, which occurs as a multimeric complex in solution, also catalyzes DNA cleavage during initiation of rolling circle replication. To identify the tomato golden mosaic virus AL1 domains that mediate protein oligomerization, DNA binding, and DNA cleavage, a series of truncated AL1 proteins were produced in a baculovirus expression system and assayed for each activity. These experiments localized the AL1 oligomerization domain between amino acids 121 and 181, the DNA binding domain between amino acids 1 and 181, and the DNA cleavage domain between amino acids 1 and 120. Deletion of the first 29 amino acids of AL1 abolished DNA binding and DNA cleavage, demonstrating that an intact N terminus is required for both activities. The observation that the DNA binding domain includes the oligomerization domain suggested that AL1-AL1 protein interaction may be a prerequisite for DNA binding but not for DNA cleavage. The significance of these results for AL1 function during geminivirus replication and transcription is discussed.}, number={15}, journal={Journal of Biological Chemistry}, author={Orozco, B. M. and Miller, A. B. and Settlage, S. B. and Hanley-Bowdoin, Linda}, year={1997}, pages={9840–9846} } @article{settlage_miller_hanley-bowdoin_1996, title={Interactions between geminivirus replication proteins}, volume={70}, number={10}, journal={Journal of Virology}, author={Settlage, S. B. and Miller, A. B. and Hanley-Bowdoin, L.}, year={1996}, pages={6790} } @article{settlage_wilson_kwanyuen_1995, title={Localization of diacylglycerol acyltransferase to oil body associated endoplasmic reticulum}, volume={33}, number={4}, journal={Plant Physiology and Biochemistry}, author={Settlage, S. B. and Wilson, R. F. and Kwanyuen, P.}, year={1995}, pages={399} }