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During spermatogenesis, multiple testis-specific histone variants are involved in the dynamic chromatin transitions. H2BFWT is a primate testis-specific H2B variant with hitherto unclear functions, and SNPs of H2BFWT are closely associated with male non-obstructive infertility. Here, we found that H2BFWT is preferentially localized in the sub-telomeric regions and the promoters of genes highly expressed in testis from differentiated spermatogonia to early spermatocytes. Cryo-EM structural analysis shows that H2BFWT nucleosomes are defined by weakened interactions between H2A-H2BFWT dimer and H4, and between histone octamer and DNA. Furthermore, one of its SNPs, H2BFWTH100R further destabilizes nucleosomes and increases the nucleosome unwrapping rate by interfering with the interaction with H4K91. Our results suggest that H2BFWT may be necessary for the regulation of spermatogenesis-related gene expression by decreasing transcriptional barriers, and that H2BFWTH100R overdrives its nucleosome-destabilizing effects which causes infertility.

Background: Structural variations (SVs) are common genetic alterations in the human genome that could cause different phenotypes and various diseases including cancer. However, the detection of structural variations using the second-generation sequencing was limited by its short read-length which in turn restrained our understanding of structural variations. Methods: In this study, we developed a 28-gene panel for long-read sequencing and employed it to both Oxford Nanopore Technologies and Pacific Biosciences platforms. We analyzed structural variations in the 28 breast cancer-related genes through long-read genomic and transcriptomic sequencing of tumor, para-tumor and blood samples in 19 breast cancer patients. Results: Our results showed that some somatic SVs were recurring among the selected genes, though the majority of them occurred in the non-exonic region. We found evidence supporting the existence of hotspot regions for SVs, which extended our previous understanding that they exist only for single nucleotide variations. Conclusions: In conclusion, we employed long-read genomic and transcriptomic sequencing in identifying SVs from breast cancer patients and proved that this approach holds great potential in clinical application.

Posttranslational modifications (PTMs) of histones represent a crucial regulatory mechanism of nucleosome and chromatin dynamics in various of DNA-based cellular processes, such as replication, transcription and DNA damage repair. Lysine succinylation (Ksucc) is a newly identified histone PTM, but its regulation and function in chromatin remain poorly understood. Here, we utilized an expressed protein ligation (EPL) strategy to synthesize histone H4 with site-specific succinylation at K77 residue (H4K77succ), an evolutionarily conserved succinylation site at the nucleosomal DNA-histone interface. We then assembled mononucleosomes with the semisynthetic H4K77succ in vitro. We demonstrated that this succinylation impacts nucleosome dynamics and promotes DNA unwrapping from the histone surface, which allows proteins such as transcription factors to rapidly access buried regions of the nucleosomal DNA. In budding yeast, a lysine-to-glutamic acid mutation, which mimics Ksucc, at the H4K77 site reduced nucleosome stability and led to defects in DNA damage repair and telomere silencing in vivo. Our findings revealed this uncharacterized histone modification has important roles in nucleosome and chromatin dynamics.

Significance Cells avoid clashes between DNA repair machineries and the transcription apparatus by temporary halting gene expression in the vicinity of DNA double-strand breaks (DSBs), an emerging DNA damage response (DDR) that underlies genome integrity protection. In this study, we screened for novel activities that may be important in this DDR and have identified the DYRK1B kinase as a component of the mammalian DDR that specializes in fine-tuning DSB repair on actively transcribed chromatin. Moreover, global analysis of DYRK1B substrates has led to the identification of the histone methyltransferase EHMT2 as a DYRK1B target and effector. Our findings uncover the DYRK1B network as a DDR subpathway that preserves the integrity of active chromatin., DNA double-strand breaks (DSBs) trigger transient pausing of nearby transcription, an emerging ATM-dependent response that suppresses chromosomal instability. We screened a chemical library designed to target the human kinome for new activities that mediate gene silencing on DSB-flanking chromatin, and have uncovered the DYRK1B kinase as an early respondent to DNA damage. We showed that DYRK1B is swiftly and transiently recruited to laser-microirradiated sites, and that genetic inactivation of DYRK1B or its kinase activity attenuated DSB-induced gene silencing and led to compromised DNA repair. Notably, global transcription shutdown alleviated DNA repair defects associated with DYRK1B loss, suggesting that DYRK1B is strictly required for DSB repair on active chromatin. We also found that DYRK1B mediates transcription silencing in part via phosphorylating and enforcing DSB accumulation of the histone methyltransferase EHMT2. Together, our findings unveil the DYRK1B signaling network as a key branch of mammalian DNA damage response circuitries, and establish the DYRK1B–EHMT2 axis as an effector that coordinates DSB repair on transcribed chromatin.

Histone variants, present in various cell types and tissues, are known to exhibit different functions. For example, histone H3.3 and H2A.Z are both involved in gene expression regulation, whereas H2A.X is a specific variant that responds to DNA double-strand breaks. In this study, we characterized H4G, a novel hominidae-specific histone H4 variant. We found that H4G is expressed in a variety of human cell lines and exhibit tumor-stage dependent overexpression in tissues from breast cancer patients. We found that H4G localized primarily to the nucleoli of the cell nucleus. This localization was controlled by the interaction of the alpha-helix 3 of the histone fold motif with a histone chaperone, nucleophosmin 1. In addition, we found that modulating H4G expression affects rRNA expression levels, protein synthesis rates and cell-cycle progression. Our data suggest that H4G expression alters nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues.

Regulation of transcription elongation is one of the key mechanisms employed to control gene expression. The single-subunit mitochondrial RNA polymerase (mtRNAP) transcribes mitochondrial genes, such as those related to ATP synthesis. We investigated how mitochondrial transcription elongation factor (TEFM) enhances mtRNAP transcription elongation using a single-molecule optical-tweezers transcription assay, which follows transcription dynamics in real time and allows the separation of pause-free elongation from transcriptional pauses. We found that TEFM enhances the stall force of mtRNAP. Although TEFM does not change the pause-free elongation rate, it enhances mtRNAP transcription elongation by reducing the frequency of long-lived pauses and shortening their durations. Furthermore, we demonstrate how mtRNAP passes through the conserved sequence block II, which is the key sequence for the switch between DNA replication and transcription in mitochondria. Our findings elucidate how both TEFM and mitochondrial genomic DNA sequences directly control the transcription elongation dynamics of mtRNAP.

Canonical histones (H2A, H2B, H3, and H4) are present in all eukaryotes where they package genomic DNA and participate in numerous cellular processes, such as transcription regulation and DNA repair. In addition to the canonical histones, there are many histone variants, which have different amino acid sequences, possess tissue-specific expression profiles, and function distinctly from the canonical counterparts. A number of histone variants, including both core histones (H2A/H2B/H3/H4) and linker histones (H1/H5), have been identified to date. Htz1 (H2A.Z) and CENP-A (CenH3) are present from yeasts to mammals, and H3.3 is present from Tetrahymena to humans. In addition to the prevalent variants, others like H3.4 (H3t), H2A.Bbd, and TH2B, as well as several H1 variants, are found to be specific to mammals. Among them, H2BFWT, H3.5, H3.X, H3.Y, and H4G are unique to primates (or Hominidae). In this review, we focus on localization and function of primate- or hominidae-specific histone variants.

The recent discovery of the cancer-associated E76K mutation in histone H2B (H2BE76-to-K) in several types of cancers revealed a new class of oncohistone. H2BE76K weakens the stability of histone octamers, alters gene expression, and promotes colony formation. However, the mechanism linking the H2BE76K mutation to cancer development remains largely unknown. In this study, we knock in the H2BE76K mutation in MDA-MB-231 breast cancer cells using CRISPR/Cas9 and show that the E76K mutant histone H2B preferentially localizes to genic regions. Interestingly, genes upregulated in the H2BE76K mutant cells are enriched for the E76K mutant H2B and are involved in cell adhesion and proliferation pathways. We focused on one H2BE76K target gene, ADAM19 (a disintegrin and metalloproteinase-domain-containing protein 19), a gene highly expressed in various human cancers including breast invasive carcinoma, and demonstrate that H2BE76K directly promotes ADAM19 transcription by facilitating efficient transcription along the gene body. ADAM19 depletion reduced the colony formation ability of the H2BE76K mutant cells, whereas wild-type MDA-MB-231 cells overexpressing ADAM19 mimics the colony formation phenotype of the H2BE76K mutant cells. Collectively, our data demonstrate the mechanism by which H2BE76K deregulates the expression of genes that control oncogenic properties through a combined effect of its specific genomic localization and nucleosome destabilization effect.

RecQL5, a mammalian RecQ family protein, is involved in the regulation of transcription elongation, DNA damage response, and DNA replication. Here, we identified and characterized an alternative splicing isoform of RECQL5 (RECQL5β1), which contains 17 additional amino acid residues within the RECQL5 KIX domain when compared with the canonical isoform (RECQL5β). RECQL5β1 had a markedly decreased binding affinity to RNA polymerase II (Pol II) and poorly competed with the transcription elongation factor TFIIS for binding to Pol II. As a result, this isoform has a weaker activity for repression of transcription elongation. In contrast, we discovered that RECQL5β1 could bind stronger to MRE11, which is a primary sensor of DNA double-strand breaks (DSBs). Furthermore, we found that RECQL5β1 promoted DNA repair in the RECQL5β1 rescue cells. These results suggest that RECQL5β mainly functions as a transcription repressor, while the newly discovered RECQL5β1 has a specialized role in DNA damage response. Taken together, our data suggest a cellular-functional specialization for each KIX splicing isoform in the cell.

The hominidae-specific histone variant H4G is expressed in breast cancer patients in a stage-dependent manner. H4G localizes primarily in the nucleoli via its interaction with nucleophosmin (NPM1). H4G is involved in rDNA transcription and ribosome biogenesis, which facilitates breast cancer cell proliferation. However, the molecular mechanism underlying this process remains unknown. Here, we show that H4G is not stably incorporated into nucleolar chromatin, even with the chaperoning assistance of NPM1. H4G likely form transient nucleosome-like-structure that undergoes rapid dissociation. In addition, the nucleolar chromatin in H4GKO cells is more compact than WT cells. Altogether, our results suggest that H4G relaxes the nucleolar chromatin and enhances rRNA transcription by forming destabilized nucleosome in breast cancer cells.

Organ formation relies on the orchestration of pattern formation, proliferation and growth during development. How these processes are integrated at the individual cell level remains unclear. In the past decades, studies using

Organ formation relies on the orchestration of pattern formation, proliferation and growth during development. How these processes are integrated at the individual cell level remains unclear. In the past decades, studies using Drosophila wing imaginal discs as a model system have provided valuable insights into pattern formation, growth control and regeneration. Here, we provide single cell transcriptomic landscapes of pattern formation, proliferation and growth of wing imaginal discs. We found that patterning information is robustly maintained in the single cell transcriptomic data and can provide reference matrices for computationally mapping single cells into discrete spatial domains. Assignment of wing disc single cells to spatial subregions facilitates examination of patterning refinement processes. We also clustered single cells into different proliferation and growth states and evaluated the correlation between cell proliferation/growth states and spatial patterning. Furthermore, single cell transcriptomic analyses allowed us to quantitatively examine disturbances of differentiation, proliferation and growth in a well-established tumor model. We provide a database to explore these datasets at http://drosophilayanlab-virtual-wingdisc.ust.hk:3838/v2/This article has an associated 'The people behind the papers' interview.

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.

Histone variants, present in various cell types and tissues, are known to exhibit different functions. For example, histone H3.3 and H2A.Z are both involved in gene expression regulation, whereas H2A.X is a specific variant that responds to DNA double-strand breaks. In this study, we characterized H4G, a novel hominidae-specific histone H4 variant. H4G expression was found in a variety of cell lines and was particularly overexpressed in the tissues of breast cancer patients. H4G was found to localize primarily to the nucleoli of the cell nucleus. This localization was controlled by the interaction of the alpha helix 3 of the histone fold motif with the histone chaperone, nucleophosphomin 1. In addition, we found that H4G nucleolar localization increased rRNA levels, protein synthesis rates, and cell cycle progression. Furthermore, micrococcal nuclease digestion of H4G-containing nucleosomes reconstitutedin vitroindicated that H4G destabilizes the nucleosome, which may serve to alter nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues.

Posttranslational modifications of histones play key roles in the dynamic regulation of chromatin structure. Lysine succinylation is a new type of histone modification, but its biological significance in chromatin structure and dynamics remains unknown. Here we develop a chemical approach to site-specifically install a succinyl lysine analog into histones. This analog serves as an ideal structural and functional mimic to natural succinyl lysine. The incorporation of this succinylation mimic into histone H2B at lysine 34, a succinylation site at the nucleosomal DNA-histone interface, leads to significant decrease in nucleosome stability in vitro, which is consistent with the defects in chromatin structure of a budding yeast strain containing a lysine-to-glutamate mutation at the corresponding residue of yeast histone H2B. This study provides a simple method for the rapid generation of histones with site-specific succinylation mimics, and reveals novel regulatory mechanisms of histone succinylation in the dynamic organization of chromatin.

Cell delamination is a conserved morphogenetic process important for the generation of cell diversity and maintenance of tissue homeostasis. Here, we used

RNA polymerase pausing represents an important mechanism of transcriptional regulation. In this study, we use a single-molecule transcription assay to investigate the effect of template base-pair composition on pausing by RNA polymerase II and the evolutionarily distinct mitochondrial polymerase Rpo41. For both enzymes, pauses are shorter and less frequent on GC-rich templates. Significantly, incubation with RNase abolishes the template dependence of pausing. A kinetic model, wherein the secondary structure of the nascent RNA poses an energetic barrier to pausing by impeding backtracking along the template, quantitatively predicts the pause densities and durations observed. The energy barriers extracted from the data correlate well with RNA folding energies obtained from cotranscriptional folding simulations. These results reveal that RNA secondary structures provide a cis -acting mechanism by which sequence modulates transcriptional elongation.

Despite the identification of H2A.Bbd as a new vertebrate-specific replacement histone variant several years ago, and despite the many in vitro structural characterizations using reconstituted chromatin complexes consisting of this variant, the existence of H2A.Bbd in the cell and its location has remained elusive. Here, we report that the native form of this variant is present in highly advanced spermiogenic fractions of mammalian testis at the time when histones are highly acetylated and being replaced by protamines. It is also present in the nucleosomal chromatin fraction of mature human sperm. The ectopically expressed non-tagged version of the protein is associated with micrococcal nuclease-refractory insoluble fractions of chromatin and in mouse (20T1/2) cell line, H2A.Bbd is enriched at the periphery of chromocenters. The exceedingly rapid evolution of this unique X-chromosome-linked histone variant is shared with other reproductive proteins including those associated with chromatin in the mature sperm (protamines) of many vertebrates. This common rate of evolution provides further support for the functional and structural involvement of this protein in male gametogenesis in mammals.

Histone variants play important roles in regulation of chromatin structure and function. To understand the structural role played by histone variants H2A.Z and H3.3, both of which are implicated in transcription regulation, we conducted extensive biochemical and biophysical analysis on mononucleosomes reconstituted from either random-sequence DNA derived from native nucleosomes or a defined DNA nucleosome positioning sequence and recombinant human histones. Using established electrophoretic and sedimentation analysis methods, we compared the properties of nucleosomes containing canonical histones and histone variants H2A.Z and H3.3 (in isolation or in combination). We find only subtle differences in the compaction and stability of the particles. Interestingly, both H2A.Z and H3.3 affect nucleosome positioning, either creating new positions or altering the relative occupancy of the existing nucleosome position space. On the other hand, only H2A.Z-containing nucleosomes exhibit altered linker histone binding. These properties could be physiologically significant as nucleosome positions and linker histone binding partly determine factor binding accessibility.

Structural variability within histone families, such as H2A, can be achieved through 2 primary mechanisms: the expression of histone variants and the incorporation of chemical modifications. The histone H2A family contains several variants in addition to the canonical H2A forms. In this review, recent developments in the study of the heteromorphous variants H2A.X, H2A.Z, and macroH2A will be discussed. Particular focus will be given to the post-translational modifications (PTMs) of these variants, including phosphorylation, ubiquitination, acetylation, and methylation. The combination of the newly identified N- and C-terminal tail PTMs expands the multiplicity of roles that the individual H2A variants can perform. It is of additional interest that analogous sites within these different histone variants can be similarly modified. Whether this is a redundant function or a finely tuned one, designed to meet specific needs, remains to be elucidated.

Replication protein A (RPA) complex has been shown, using both in vivo and in vitro approaches, to be required for most aspects of eukaryotic DNA metabolism: replication, repair, telomere maintenance and homologous recombination. Here, we review recent data concerning the function and biological importance of the multi-RPA complex. There are distinct complexes of RPA found in the biological kingdoms, although for a long time only one type of RPA complex was believed to be present in eukaryotes. Each complex probably serves a different role. In higher plants, three distinct large and medium subunits are present, but only one species of the smallest subunit. Each of these protein subunits forms stable complexes with their respective partners. They are paralogs as complex. Humans possess two paralogs and one analog of RPA. The multi-RPA system can be regarded as universal in eukaryotes. Among eukaryotic kingdoms, paralogs, orthologs, analogs and heterologs of many DNA synthesis-related factors, including RPA, are ubiquitous. Convergent evolution seems to be ubiquitous in these processes. Using recent findings, we review the composition and biological functions of RPA complexes.

Molecular evolutionary analyses revealed that histone H2A.Bbd is a highly variable quickly evolving mammalian replacement histone variant, in striking contrast to all other histones. At the nucleotide level, this variability appears to be the result of a larger amount of nonsynonymous variation, which affects to a lesser extent, the structural domain of the protein comprising the histone fold. The resulting amino acid sequence diversity can be predicted to affect the internucleosomal and intranucleosomal histone interactions. Our phylogenetic analysis has allowed us to identify several of the residues involved. The biophysical characterization of nucleosomes reconstituted with recombinant mouse H2A.Bbd and their comparison to similar data obtained with human H2A.Bbd clearly support this notion. Despite the high interspecific amino acid sequence variability, all of the H2A.Bbd variants exert similar structural effects at the nucleosome level, which result in an unfolded highly unstable nucleoprotein complex. Such structure resembles that previously described for the highly dynamically acetylated nucleosomes associated with transcriptionally active regions of the genome. Nevertheless, the structure of nucleosome core particles reconstituted from H2A.Bbd is not affected by the presence of a hyperacetylated histone complement. This suggests that replacement by H2A.Bbd provides an alternative mechanism to unfold chromatin structure, possibly in euchromatic regions, in a way that is not dependent on acetylation.

The human UV-damaged DNA binding protein (UV-DDB), a heterodimeric protein composed of 127 kDa (UV-DDB1) and 48 kDa (UV-DDB2) subunits, has been shown to be involved in DNA repair. To elucidate the in vivo function of plant UV-DDB2, we have analyzed T-DNA insertion mutants of the Arabidopsis thaliana UV-DDB2 subunit (atuv-ddb2 mutants) and AtUV-DDB2 RNAi silenced plants (atuv-ddb2 silenced plants). atuv-ddb2 mutants and atuv-ddb2 silenced plants were both viable, suggesting that AtUV-DDB2 is not essential for survival. Interestingly, both plant types showed a dwarf phenotype, implying impaired growth of the meristem. To the best of our knowledge, this is the first occasion that a dwarf phenotype has been found to be associated with a UV-DDB2 mutation in either plants or animals. The mutants also demonstrated increased sensitivity to UV irradiation, methyl methanesulfonate and hydrogen peroxide treatment, indicating that AtUV-DDB2 is also involved in DNA repair. Our results lead us to suggest that not only does AtUV-DDB2 function in DNA repair, it also has a direct or indirect influence on cell proliferation in the plant meristem.

Understanding the mechanisms responsible for UV-B irradiation-induced DNA damage repair in plants is important for agricultural technology in that it will potentially enable the development of plants with enhanced growth rates and crop yields. Unlike yeast and mammalian cells, intact plants use sunlight for photosynthesis and are thus chronically exposed to the UV band wavelengths present in solar radiation. UV induces DNA damage, which can be corrected by DNA repair mechanisms such as photoreactivation and excision repair. Recently, details of several DNA repair mechanisms have become clear in plants. We made transgenic rice overexpressing genes involved in excision repair or plant-specific DNA repair, and measured their tolerance to UV-B. We found that OsUV-DDB2 and OsSEND-1 transgenic lines had higher tolerance to UV-B than the wild type. In this review, recent advances in understanding repair of DNA damage from UV-B radiation in plants and the prospects for the development of UV resistant plants are discussed.

A novel RecA-like protein, differing from Dmc1 and Rad51, was characterized in Oryza sativa L. cv. Nipponbare. Because the protein is homologous to bacterial RadA, the gene was designated OsRadA . The open reading frame was predicted to encode a 66 kDa protein of 619 amino acid residues and was found in plants but not animals or yeast. OsRadA showed D-loop and single-stranded DNA-dependent ATPase activities. Gene expression was found to be high in meristematic tissues, and was localized in the nucleus. An RNAi mutant of Arabidopsis thaliana RadA ( AtRadA ) was sensitive to mutagenic agents such as UV and MMC, suggesting that RadA functions in DNA repair.

Previously, we described a novel DNA polymerase, designated as OsPolI-like, from rice. The OsPolI-like showed a high degree of sequence homology with the DNA polymerase I of cyanobacteria and was localized in the plastid. Here, we describe two PolI-like polymerases, designated as AtPolI-like A and AtPolI-like B, from Arabidopsis thaliana. In situ hybridization analysis demonstrated expression of both mRNAs in proliferating tissues such as the shoot apical meristem. Analysis of the localizations of GFP fusion proteins showed that AtPolI-like A and AtPolI-like B were localized to plastids. AtPolI-like B expression could be induced by exposure to the mutagen H{sub 2}O{sub 2}. These results suggested that AtPolI-like B has a role in the repair of oxidation-induced DNA damage. Our data indicate that higher plants possess two plastid DNA polymerases that are not found in animals and yeasts.

The origin recognition complex (ORC) protein plays a critical role in DNA replication through binding to sites (origins) where replication commences. The protein is composed of six subunits (ORC1 to 6) in animals and yeasts. Our knowledge of the ORC protein in plants is, however, much less complete. We have performed cDNA cloning and characterization of ORC subunits in rice (Oryza sativa L. cv. Nipponbare) in order to facilitate study of plant DNA replication mechanisms. Our previous report provided a description of a gene, ORC1 (OsORC1), that encodes one of the protein subunits. The present report extends this initial analysis to include the genes that encode four other rice ORC subunits, OsORC2, 3, 4 and 5. Northern hybridization analyses demonstrated the presence of abundant transcripts for all OsORC subunits in shoot apical meristems (SAM) and cultured cells, but not in mature leaves. Interestingly, only OsORC5 showed high levels of expression in organs in which cell proliferation is not active, such as flag leaves, the ears and the non-tip roots. The pattern of expression of OsORC2 also differed from other OsORC subunits. When cell proliferation was temporarily halted for 6-10 days by removal of sucrose from the growth medium, expression of OsORC1, OsORC3, OsORC4 and OsORC5 was substantially reduced. However, the level of expression of OsORC2 remained constant. We suggest from these results that expression of OsORC1, 3, 4 and 5 are correlated with cell proliferation, but the expression of OsORC2 is not.

Proliferating cell nuclear antigen (PCNA) is an essential protein for both DNA replication and DNA repair. In the present study using two-hybrid analysis with PCNA from rice, Oryza sativa L. cv. Nipponbare (OsPCNA), we found that OsPCNA interacted with rice DnaJ protein. We have identified DnaJ and designated it as OsDnaJ. OsDnaJ was able to bind to OsPCNA in vitro. Transcripts of OsDnaJ were found to be strongly expressed in the proliferating cells. mRNA of DnaJ was induced by UV and DNA-damaging agents such as H2O2. The expression patterns of OsPCNA were almost the same as OsDnaJ. The relationship between OsPCNA and OsDnaJ is discussed.

COP10 is a ubiquitin-conjugating enzyme variant (UEV), which is thought to act together with COP1, DET1, and the COP9 signalosome (CSN) in Arabidopsis to repress photomorphogenesis. Here, we demonstrate that COP10 interacts with ubiquitin-conjugating enzymes (E2s) in vivo, and can enhance their activity in vitro, an activity distinct from previous characterized UEVs such as MMS2 and UEV1. Furthermore, we show that COP10 forms a complex with UV-damaged DNA-binding protein 1a (DDB1a) and de-etiolated 1 (DET1), and physically interacts with COP1 and the CSN. Purified CDD (COP10, DDB1, DET1) complex also shows enhancement of E2 activity (UEA) similar to that observed with COP10 itself. Our data suggests that COP10, along with COP1 and the CSN, promotes the degradation of positive regulators of photomorphogenesis, such as the transcription factor HY5, via the ubiquitin/26S proteasome system. Thus, the CDD complex may act as a ubiquitylation-promoting factor to regulate photomorphogenesis.

Little is known about the functions of DNA polymerase lambda (Pol lambda) recently identified in mammals. From the genomic sequence information of rice and Arabidopsis, we found that Pol lambda may be the only member of the X-family in higher plants. We have succeeded in isolating the cDNA and recombinant protein of Pol lambda in a higher plant, rice (Oryza sativa L. cv. Nipponbare) (OsPol lambda). OsPol lambda had activities of DNA polymerase, terminal deoxyribonucleotidyl transferase and deoxyribose phosphate lyase, a marker enzyme for base excision repair. It also interacted with rice proliferating cell nuclear antigen (OsPCNA) in a pull-down assay. OsPCNA increased the processivity of OsPol lambda. Northern blot analysis showed that the level of OsPol lambda expression correlated with cell proliferation in meristematic and meiotic tissues, and was induced by DNA-damaging treatments. These properties suggest that plant Pol lambda is a DNA repair enzyme which functions in plant meristematic and meiotic tissues, and that it can substitute for Pol beta and terminal deoxyribonucleotidyl transferase.

To study whether metabolic control of proliferating cell nuclear antigen (PCNA) during the cell cycle is similar to that of associated protein factors, two-hybrid analysis with PCNA from rice (Oryza sativa L. cv. Nipponbare) was performed. PCNA interacted with rice Rpt6, which is the ATPase subunit of 26S proteasome, both in vitro and in vivo, and the degradation of PCNA was disrupted by the proteasome in vivo. The tissue-specific expression pattern of the transcripts of Rpt6 and PCNA suggested that the rice proteasome played important roles in DNA replication involving PCNA. These findings indicate a proteasome-dependent degradation of PCNA.

DNA replication is a process that is highly conserved among eukaryotes. Nonetheless, little is known about the proteins involved in it in plants. Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding protein with several functions in DNA metabolism in humans and yeast and supposedly also in plants. Here we report on the regulation of OsRPA2, the 32-kDa subunit of RPA from rice ( Oryza sativa L.). We found conserved regulation mechanisms at the level of gene expression between animal and plant RPA2 genes and distinct features of OsRPA2 regulation at the level of protein expression. Unlike in animals or in yeast, protein abundance in rice was regulated in a cell cycle phase-specific manner and was altered after UV-C light exposure. On the other hand, posttranslational modification through phosphorylation did not appear to play a pivotal role in rice as it does in animal cells. Our results indicate that plant-specific mechanisms of regulation have evolved for RPA2 within the generally well-conserved process of DNA replication, suggesting specific requirements for regulation of DNA metabolism in plants as compared to other eukaryotes.

The eukaryotic polymerase processivity factor, proliferating cell nuclear antigen (PCNA), interacts with many cell cycle-regulator proteins and with proteins involved in the mechanisms of DNA replication and repair. In the present study using two-hybrid analysis with PCNA from rice, Oryza sativa L. cv. Nipponbare (OsPCNA), we found that OsPCNA interacted in vitro and in vivo with rice JUN-activation-domain-binding protein 1 (OsJab1), which is known as COP9/signalsome subunit 5. Both OsPCNA and OsJab1 transcripts were expressed strongly in the shoot apical meristem and weakly in young leaves, flag leaves, ears, roots and root tips. No expression was detected in the mature leaves. The OsPCNA and OsJab1 proteins were expressed and accumulated mostly in the shoot apical meristem and ears, suggesting that OsJab1 is involved in cell proliferation in cooperation with OsPCNA. The role of OsPCNA with OsJab1 in plant DNA proliferation is discussed.

A novel endonuclease, a new member of the RAD2 nuclease family, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare), and designated as OsSEND-1. The open reading frame of the OsSEND-1 cDNA encoded a predicted product of 641 amino acid residues with a molecular weight of 69.9 kDa. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nuclease family proteins, especially RAD2 nuclease, but it differed markedly from FEN-1, XPG or HEX1/EXO1. The N- and I-domains in the family were highly conserved in the OsSEND-1 sequence. The protein was much smaller than XPG, but larger than HEX1/EXO1 and FEN-1. The genome sequence was composed of 14 exons, and was localized at the almost terminal region of the short arm of chromosome 8. Northern blotting and in situ hybridization analyses demonstrated preferential expression of OsSEND-1 mRNA in proliferating tissues such as meristem. The mRNA level of OsSEND-1 was induced by UV and DNA-damaging agent such as MMS or H2O2, indicating that OsSEND-1 has some roles in the repair of many types of damaged DNA. The recombinant peptide showed endonuclease activity.

DNA polymerase delta (pol delta), which is comprised of at least two essential subunits, is an important enzyme involved in DNA replication and repair. We have cloned and characterized both the catalytic and small subunits of pol delta from rice (Oryza sativa L. cv. Nipponbare). The open reading frames of OsPoldelta1 and delta2 encoded a predicted product of 1105 amino acid residues with a molecular weight of 124 kDa for OsPoldelta1, and of 429 residues with a molecular weight of 48 kDa for OsPoldelta2. Northern blotting analysis indicated that OsPoldelta1 and delta2 transcripts were expressed strongly in proliferating tissues such as shoot apical meristem. The expression patterns of both subunits in the organs were slightly different. Therefore, we analyzed the spatial distribution pattern of OsPoldelta1 transcripts by in situ hybridization. In the shoot apex, OsPoldelta1 mRNA was abundant in the shoot apical meristem. In the roots, the OsPoldelta1 transcript accumulated at high levels in the root apical meristem. In mature leaves, OsPoldelta1 was induced after UV irradiation, but OsPoldelta2 was not. The amounts of the OsPoldelta1 and delta2 mRNAs in the rice cells changed rapidly during cell proliferation. These results indicated that the levels of OsPoldelta expression are markedly correlated with cell proliferation, and that some of OsPoldelta might have special roles in the leaves.

Cell delamination is a conserved morphogenetic process important for the generation of cell diversity and maintenance of tissue homeostasis. Here, we used Drosophila embryonic neuroblasts as a model to study the apical constriction process during cell delamination. We observe dynamic myosin signals both around the cell adherens junctions and underneath the cell apical surface in the neuroectoderm. On the cell apical cortex, the nonjunctional myosin forms flows and pulses, which are termed medial myosin pulses. Quantitative differences in medial myosin pulse intensity and frequency are crucial to distinguish delaminating neuroblasts from their neighbors. Inhibition of medial myosin pulses blocks delamination. The fate of a neuroblast is set apart from that of its neighbors by Notch signaling-mediated lateral inhibition. When we inhibit Notch signaling activity in the embryo, we observe that small clusters of cells undergo apical constriction and display an abnormal apical myosin pattern. Together, these results demonstrate that a contractile actomyosin network across the apical cell surface is organized to drive apical constriction in delaminating neuroblasts.

Transcription represents the first step in gene expression. It is therefore not surprising that transcription is a highly regulated process and its control is essential to understand the flow and processing of information required by the cell to maintain its homeostasis. During transcription, a DNA molecule is copied into RNA molecules that are then used to translate the genetic information into proteins; this logical pattern has been conserved throughout all three kingdoms of life, from Archaea to Eukarya, making it an essential and fundamental cellular process. Even though some viruses that encode their genome in an RNA molecule use it as a template to make mRNA, others synthesize an intermediate DNA molecule from the RNA, a process known as reverse transcription, from which regular transcription of viral genes can then proceed in the host cells.

Significance Regulation of gene expression controls fundamental cellular processes, such as growth and differentiation. Gene expression begins at transcription, in which the eukaryotic RNA polymerase (Pol II) synthesizes the precursors of mRNA during the elongation phase. The speed and fidelity of the process are regulated by many transcription elongation factors, including transcription factors IIS (TFIIS) and IIF (TFIIF), which directly interact with the enzyme. Using a single-molecule optical-tweezers assay, we have quantified the effects of these factors on the dynamics of transcription elongation by Pol II on both bare and nucleosomal DNA. We showed that TFIIF mainly prevents Pol II from pause entering, whereas TFIIS assists with pause recovery, and quantitatively described these effects on the kinetics of transcription elongation by Pol II.

Replication protein A (RPA), which is comprised of three subunits, is an important factor involved in DNA replication, repair, and transcription. We isolated and characterized 70 and 32 kDa subunits of RPA from rice (Oryza sativa cv. Nipponbare) termed OsRPA70a and OsRPA32. OsRPA70a shows a low level of homology with OsRPA1 which was isolated from deepwater rice (Oryza sativa cv. Pin Gaew 56), previously. We also succeeded to isolate OsRPA70b which is homologue to OsRPA1 from Oryza sativa cv. Nipponbare. OsRPA70a shows only 33.8% sequence identity with OsRPA70b, indicating that two different types of 70 kDa RPA subunits are present in Oryza sativa cv. Nipponbare. These subunits showed differences in their expression patterns among tissues. The transcripts of OsRPA70a and OsRPA32 were expressed strongly in proliferating tissues such as root tips and young leaves that contain root apical meristem and marginal meristem, respectively, and weakly in the mature leaves which have no proliferating tissues. On the other hand, OsRPA70b was expressed mostly in the proliferating tissues. The roles of these molecules in plant DNA replication and DNA repair are discussed.

We have isolated a 1.47 kb cDNA from rice (Oryza sativa) encoding plant homologue of a small subunit of Replication Factor C (RFC), and designated OsRFC3. The open reading frame of OsRFC3 encoded a predicted product of 367 amino acid residues with a molecular mass of 40.3 kDa. Genomic analysis indicated that the subunit existed as a single copy per genome. The transcript was highly expressed in root tips that contain root apical meristem, and the young and mature leaves. The mRNA level of OsRFC3 was significantly reduced when the cell proliferation was temporarily halted by removal of sucrose from the growth medium. When the growth-halted cells began to re-grow following addition of sucrose to the medium, OsRFC3 was again expressed at high level. The roles of OsRFC3 in plant DNA replication is discussed.

By using the rice EST database, we have isolated a 2.8 kb cDNA, termed Oryza sativa ORC1 (OsORC1), from rice (O. sativa) encoding a protein that shows homology with the eukaryotic ORC1 proteins. Alignment of the OsORC1 protein sequence with the sequence of ORC1 from human and yeasts S. cerevisiae and S. pombe showed a high degree of sequence homology (38.7, 32.9 and 35.0% identity, respectively), particularly around the C-terminal region containing the CDC-NTP domain. Interestingly, the OsORC1 protein had an A+T hook-like motif, which was not present in the human or yeast genes. Genomic analysis indicated that OsORC1 existed as a single copy per genome. OsORC1 transcripts were expressed strongly in root tips and weakly in young leaves containing root apical meristem and marginal meristem, respectively. No expression was detected in the mature leaves. The level of OsORC1 expression was significantly reduced when cell proliferation was temporarily halted by the removal of sucrose from the growth medium. When the growth-halted cells began to re-grow following addition of sucrose to the medium, OsORC1 was again expressed at high levels. These results suggested that OsORC1 is required for cell proliferation. The role of OsORC1 in plant DNA replication will be discussed.