Siripan Limsirichaikul

Three DNA Polymerases, Recruited by Different Mechanisms, Carry Out NER Repair Synthesis in Human Cells

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both pol kappa and pol delta, and both polymerases can be recovered in the same repair complexes. Pol kappa is recruited to repair sites by ubiquitinated PCNA and XRCC1 and pol delta by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on pol epsilon, recruitment of which is dependent on the alternative clamp loader CTF18-RFC.

Palm Mutants in DNA Polymerases α and η Alter DNA Replication Fidelity and Translesion Activity

ABSTRACT We isolated active mutants in Saccharomyces cerevisiae DNA polymerase α that were associated with a defect in error discrimination. Among them, L868F DNA polymerase α has a spontaneous error frequency of 3 in 100 nucleotides and 570-fold lower replication fidelity than wild-type (WT) polymerase α. In vivo, mutant DNA polymerases confer a mutator phenotype and are synergistic with msh2 or msh6, suggesting that DNA polymerase α-dependent replication errors are recognized and repaired by mismatch repair. In vitro, L868F DNA polymerase α catalyzes efficient bypass of a cis-syn cyclobutane pyrimidine dimer, extending the 3′ T 26,000-fold more efficiently than the WT. Phe34 is equivalent to residue Leu868 in translesion DNA polymerase η, and the F34L mutant of S. cerevisiae DNA polymerase η has reduced translesion DNA synthesis activity in vitro. These data suggest that high-fidelity DNA synthesis by DNA polymerase α is required for genomic stability in yeast. The data also suggest that the phenylalanine and leucine residues in translesion and replicative DNA polymerases, respectively, might have played a role in the functional evolution of these enzyme classes.

Identification of the first ATRIP-deficient patient and novel mutations in ATR define a clinical spectrum for ATR-ATRIP Seckel Syndrome

A homozygous mutational change in the Ataxia-Telangiectasia and RAD3 related (ATR) gene was previously reported in two related families displaying Seckel Syndrome (SS). Here, we provide the first identification of a Seckel Syndrome patient with mutations in ATRIP, the gene encoding ATR–Interacting Protein (ATRIP), the partner protein of ATR required for ATR stability and recruitment to the site of DNA damage. The patient has compound heterozygous mutations in ATRIP resulting in reduced ATRIP and ATR expression. A nonsense mutational change in one ATRIP allele results in a C-terminal truncated protein, which impairs ATR–ATRIP interaction; the other allele is abnormally spliced. We additionally describe two further unrelated patients native to the UK with the same novel, heterozygous mutations in ATR, which cause dramatically reduced ATR expression. All patient-derived cells showed defective DNA damage responses that can be attributed to impaired ATR–ATRIP function. Seckel Syndrome is characterised by microcephaly and growth delay, features also displayed by several related disorders including Majewski (microcephalic) osteodysplastic primordial dwarfism (MOPD) type II and Meier-Gorlin Syndrome (MGS). The identification of an ATRIP–deficient patient provides a novel genetic defect for Seckel Syndrome. Coupled with the identification of further ATR–deficient patients, our findings allow a spectrum of clinical features that can be ascribed to the ATR–ATRIP deficient sub-class of Seckel Syndrome. ATR–ATRIP patients are characterised by extremely severe microcephaly and growth delay, microtia (small ears), micrognathia (small and receding chin), and dental crowding. While aberrant bone development was mild in the original ATR–SS patient, some of the patients described here display skeletal abnormalities including, in one patient, small patellae, a feature characteristically observed in Meier-Gorlin Syndrome. Collectively, our analysis exposes an overlapping clinical manifestation between the disorders but allows an expanded spectrum of clinical features for ATR–ATRIP Seckel Syndrome to be defined.

Geraniinic acid derivative from the leaves of Phyllanthus reticulatus

Chemical constituents as well as cytotoxic and insecticidal activity of the crude methanol extract from the leaves of Phyllanthus reticulatus Poir. (Euphorbiaceae) were investigated. (5R*,6R*)-4,6-Dimethoxycarbonyl-5-[2′,3′,4′-trihydroxy-6′-(methoxycarbonyl) phenyl]-5,6-dihydro-2H-pyran-2-one (1) along with 3,4,3′-tri-O-methylellagic acid, and methyl gallate were isolated from the dichloromethane extract. Determination of their structures was based on spectroscopic analysis. Compound 1 possessed a very weak insecticidal activity against Spodoptera frugiperda (Sf9) with an IC50 value of 27.27 μg/mL.

Genome-wide analysis reveals phytohormone action during cassava storage root initiation.

Development of storage roots is a process associated with a phase change from cell division and elongation to radial growth and accumulation of massive amounts of reserve substances such as starch. Knowledge of the regulation of cassava storage root formation has accumulated over time; however, gene regulation during the initiation and early stage of storage root development is still poorly understood. In this study, transcription profiling of fibrous, intermediate and storage roots at eight weeks old were investigated using a 60-mer-oligo microarray. Transcription and gene expression were found to be the key regulating processes during the transition stage from fibrous to intermediate roots, while homeostasis and signal transduction influenced regulation from intermediate roots to storage roots. Clustering analysis of significant genes and transcription factors (TF) indicated that a number of phytohormone-related TF were differentially expressed; therefore, phytohormone-related genes were assembled into a network of correlative nodes. We propose a model showing the relationship between KNOX1 and phytohormones during storage root initiation. Exogeneous treatment of phytohormones N6-benzylaminopurine and 1-Naphthaleneacetic acid were used to induce the storage root initiation stage and to investigate expression patterns of the genes involved in storage root initiation. The results support the hypothesis that phytohormones are acting in concert to regulate the onset of cassava storage root development. Moreover, MeAGL20 is a factor that might play an important role at the onset of storage root initiation when the root tip becomes swollen.

Comparison of In Vitro Binding of Bile Salt by Pectins from Various Sources

Binding of bile salts by dietary fiber is believed to promote their excretion and hence to reduce the serum cholesterol level in man and experimental animals. In this study, the binding efficiency of soluble pectin from various sources, i.e., apple, citrus and pomelo, was examined. Sodium deoxycholate and sodium cholate hydrate were used as a model to represent bile salt in human body. The binding efficiency was assayed by acid reaction, thin layer chromatography (TLC) and enzyme cycling method. The results demonstrated that enzyme cycling method was the most suitable for assaying the in-vitro binding of bile salts while the TLC was not very sensitive, i.e., low amount of bile salts cannot be detected by TLC. Excess pectin from binding test could also interfere the acid reaction method even though the centrifugation was used to remove the excess pectin. When the concentration of pectin was increased, the binding efficiency with sodium deoxycholate increased. However, at 1% w/w of pectin, the binding efficiency decreased. The exception is for pomelo pectin in which the binding efficiency increased when the pectin concentration increased. With sodium cholate hydrate, only slight difference in binding efficiency was observed for all types and concentrations of pectin. The results indicate that the ability to bind bile salts of pectin might be responsible for its hypocholesterolemic action observed in experimental animals and humans.

Hyper-Phosphorylated Retinoblastoma Protein Suppresses Telomere Elongation

Immortalized cell lines maintain telomeres by the expression of telomerase or by a mechanism designated alternative lengthening of telomeres (ALT). Although DNA polymerase α (pol-α) is reported to be required for telomere maintenance, the critical role of pol-α in telomere maintenance has not been firmly determined. We examined the role of retinoblastoma protein (pRb) and pol-α in the regulation of telomere length, using telomere-fiber FISH. Telomere length varied dependent on the intracellular abundance of pol-α or pRb in HeLa cells. A proportion of hyper-phosphorylated pRb (ppRb) molecules localized to sites of telomeric DNA replication in HeLa cells. Pol-α might thus contribute to telomere maintenance, and might be regulated by ppRb.

3D-structured illumination microscopy reveals clustered DNA double-strand break formation in widespread γH2AX foci after high LET heavy-ion particle radiation

DNA double-strand breaks (DSBs) induced by ionising radiation are considered the major cause of genotoxic mutations and cell death. While DSBs are dispersed throughout chromatin after X-rays or γ-irradiation, multiple types of DNA damage including DSBs, single-strand breaks and base damage can be generated within 1–2 helical DNA turns, defined as a complex DNA lesion, after high Linear Energy Transfer (LET) particle irradiation. In addition to the formation of complex DNA lesions, recent evidence suggests that multiple DSBs can be closely generated along the tracks of high LET particle irradiation. Herein, by using three dimensional (3D)-structured illumination microscopy, we identified the formation of 3D widespread γH2AX foci after high LET carbon-ion irradiation. The large γH2AX foci in G2-phase cells encompassed multiple foci of replication protein A (RPA), a marker of DSBs undergoing resection during homologous recombination. Furthermore, we demonstrated by 3D analysis that the distance between two individual RPA foci within γH2AX foci was approximately 700 nm. Together, our findings suggest that high LET heavy-ion particles induce clustered DSB formation on a scale of approximately 1 μm3. These closely localised DSBs are considered to be a risk for the formation of chromosomal rearrangement after heavy-ion irradiation.

Identification of DNA double strand breaks at chromosome boundaries along the track of particle irradiation.

Chromosomal translocations arise from misrejoining of DNA double strand breaks (DSBs) between loci located on two chromosomes. One current model suggests that spatial proximity of potential chromosomal translocation partners influences translocation probability. Ionizing radiation (IR) is a potent inducer of translocations. Accumulating evidence demonstrates that particle irradiation more frequently causes translocations compared with X‐ray irradiation. This observation has led to the hypothesis that the high frequency of translocations after particle irradiation may be due to the formation of DSBs at chromosome boundaries along the particle track, because such DSBs can be misrejoined between distinct chromosomes. In this study, we simultaneously visualized the site of IR‐induced DSBs and chromosome position by combining Immunofluorescence and fluorescence in situ hybridization. Importantly, the frequency of γH2AX foci at the chromosome boundary of chromosome 1 after carbon‐ion irradiation was >4‐fold higher than that after X‐ray irradiation. This observation is consistent with the idea that particle irradiation generates DSBs at the boundaries of two chromosomes along the track. Further, we showed that resolution of γH2AX foci at chromosome boundaries is prevented by inhibition of DNA‐PKcs activity, indicating that the DSB repair is NHEJ‐dependent. Finally, we found that γH2AX foci at chromosome boundaries after carbon‐ion irradiation contain DSBs undergoing DNA‐end resection, which promotes repair utilizing microhomology mediated end‐joining during translocation. Taken together, our study suggests that the frequency of DSB formation at chromosome boundaries is associated with the incidence of chromosomal translocations, supporting the notion that the spatial proximity between breaks is an important factor in translocation formation.