GPRC5D-Targeted CAR T Cells for Myeloma.

BACKGROUND: B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell therapies have generated responses in patients with advanced myeloma, but relapses are common. G protein-coupled receptor, class C, group 5, member D (GPRC5D) has been identified as an immunotherapeutic target in multiple myeloma. Preclinical studies have shown the efficacy of GPRC5D-targeted CAR T cells, including activity in a BCMA antigen escape model. METHODS: In this phase 1 dose-escalation study, we administered a GPRC5D-targeted CAR T-cell therapy (MCARH109) at four dose levels to patients with heavily pretreated multiple myeloma, including patients with relapse after BCMA CAR T-cell therapy. RESULTS: A total of 17 patients were enrolled and received MCARH109 therapy. The maximum tolerated dose was identified at 150×106 CAR T cells. At the 450×106 CAR T-cell dose, 1 patient had grade 4 cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome (ICANS), and 2 patients had a grade 3 cerebellar disorder of unclear cause. No cerebellar disorder, ICANS of any grade, or cytokine release syndrome of grade 3 or higher occurred in the 12 patients who received doses of 25×106 to 150×106 cells. A response was reported in 71% of the patients in the entire cohort and in 58% of those who received doses of 25×106 to 150×106 cells. The patients who had a response included those who had received previous BCMA therapies; responses were observed in 7 of 10 such patients in the entire cohort and in 3 of 6 such patients who received 25×106 to 150×106 cells. CONCLUSIONS: The results of this study of a GPRC5D-targeted CAR T-cell therapy (MCARH109) confirm that GPRC5D is an active immunotherapeutic target in multiple myeloma. (Funded by Juno Therapeutics/Bristol Myers Squibb; ClinicalTrials.gov number, NCT04555551.).

Preclinical Efficacy and Safety of a Human Embryonic Stem Cell-Derived Midbrain Dopamine Progenitor Product, MSK-DA01.

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial.

Inherited GINS1 deficiency underlies growth retardation along with neutropenia and NK cell deficiency.

Inborn errors of DNA repair or replication underlie a variety of clinical phenotypes. We studied 5 patients from 4 kindreds, all of whom displayed intrauterine growth retardation, chronic neutropenia, and NK cell deficiency. Four of the 5 patients also had postnatal growth retardation. The association of neutropenia and NK cell deficiency, which is unusual among primary immunodeficiencies and bone marrow failures, was due to a blockade in the bone marrow and was mildly symptomatic. We discovered compound heterozygous rare mutations in Go-Ichi-Ni-San (GINS) complex subunit 1 (GINS1, also known as PSF1) in the 5 patients. The GINS complex is essential for eukaryotic DNA replication, and homozygous null mutations of GINS component-encoding genes are embryonic lethal in mice. The patients' fibroblasts displayed impaired GINS complex assembly, basal replication stress, impaired checkpoint signaling, defective cell cycle control, and genomic instability, which was rescued by WT GINS1. The residual levels of GINS1 activity reached 3% to 16% in patients' cells, depending on their GINS1 genotype, and correlated with the severity of growth retardation and the in vitro cellular phenotype. The levels of GINS1 activity did not influence the immunological phenotype, which was uniform. Autosomal recessive, partial GINS1 deficiency impairs DNA replication and underlies intra-uterine (and postnatal) growth retardation, chronic neutropenia, and NK cell deficiency.

Interaction between human Ctf4 and the Cdc45/Mcm2-7/GINS (CMG) replicative helicase.

Chromosome transmission fidelity 4 (Ctf4) is a conserved protein required for DNA replication. In this report, interactions between human Ctf4 (hCtf4) and the replicative helicase containing the cell division cycle 45 (Cdc45)/minichromosome maintenance 2-7 (Mcm2-7)/Go, Ichi, Nii, and San (GINS) (CMG) proteins [human CMG (hCMG) complex] were examined. The hCtf4-CMG complex was isolated following in vitro interaction of purified proteins (hCtf4 plus the hCMG complex), coinfection of Spodoptera frugiperda (Sf9) insect cells with viruses expressing the hCMG complex and hCtf4, and from HeLa cell chromatin after benzonase and immunoprecipitation steps. The stability of the hCtf4-CMG complex depends upon interactions between hCtf4 and multiple components of the hCMG complex. The hCtf4-CMG complex, like the hCMG complex, contains DNA helicase activity that is more salt-resistant than the helicase activity of the hCMG complex. We demonstrate that the hCtf4-CMG complex contains a homodimeric hCtf4 and a monomeric hCMG complex and suggest that the homodimeric hCtf4 acts as a platform linking polymerase α to the hCMG complex. The role of the hCMG complex as the core of the replisome is also discussed.

In vitro loading of human cohesin on DNA by the human Scc2-Scc4 loader complex.

The loading of cohesin onto chromatin requires the heterodimeric complex sister chromatid cohesion (Scc)2 and Scc4 (Scc2/4), which is highly conserved in all species. Here, we describe the purification of the human (h)-Scc2/4 and show that it interacts with h-cohesin and the heterodimeric Smc1-Smc3 complex but not with the Smc1 or Smc3 subunit alone. We demonstrate that both h-Scc2/4 and h-cohesin are loaded onto dsDNA containing the prereplication complex (pre-RC) generated in vitro by Xenopus high-speed soluble extracts. The addition of geminin, which blocks pre-RC formation, prevents the loading of Scc2/4 and cohesin. Xenopus extracts depleted of endogenous Scc2/4 with specific antibodies, although able to form pre-RCs, did not support cohesin loading unless supplemented with purified h-Scc2/4. The results presented here indicate that the Xenopus or h-Scc2/4 complex supports the loading of Xenopus and/or h-cohesin onto pre-RCs formed by Xenopus high-speed extracts. We show that cohesin loaded onto pre-RCs either by h-Scc2/4 and/or the Xenopus complex was dissociated from chromatin by low salt extraction, similar to cohesin loaded onto chromatin in G(1) by HeLa cells in vivo. Replication of cohesin-loaded DNA, both in vitro and in vivo, markedly increased the stability of cohesin associated with DNA. Collectively, these in vitro findings partly recapitulate the in vivo pathway by which sister chromatids are linked together, leading to cohesion.

Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase.

The eukaryotic replicative DNA helicase, CMG, unwinds DNA by an unknown mechanism. In some models, CMG encircles and translocates along one strand of DNA while excluding the other strand. In others, CMG encircles and translocates along duplex DNA. To distinguish between these models, replisomes were confronted with strand-specific DNA roadblocks in Xenopus egg extracts. An ssDNA translocase should stall at an obstruction on the translocation strand but not the excluded strand, whereas a dsDNA translocase should stall at obstructions on either strand. We found that replisomes bypass large roadblocks on the lagging strand template much more readily than on the leading strand template. Our results indicate that CMG is a 3' to 5' ssDNA translocase, consistent with unwinding via "steric exclusion." Given that MCM2-7 encircles dsDNA in G1, the data imply that formation of CMG in S phase involves remodeling of MCM2-7 from a dsDNA to a ssDNA binding mode.

Studies on human DNA polymerase epsilon and GINS complex and their role in DNA replication.

In eukaryotic cells, DNA replication is carried out by the coordinated action of three DNA polymerases (Pols), Pol α, δ, and ε. In this report, we describe the reconstitution of the human four-subunit Pol ε and characterization of its catalytic properties in comparison with Pol α and Pol δ. Human Pol ε holoenzyme is a monomeric complex containing stoichiometric subunit levels of p261/Pol 2, p59, p17, and p12. We show that the Pol ε p261 N-terminal catalytic domain is solely responsible for its ability to catalyze DNA synthesis. Importantly, human Pol (hPol) ε was found more processive than hPol δ in supporting proliferating cell nuclear antigen-dependent elongation of DNA chains, which is in keeping with proposed roles for hPol ε and hPol δ in the replication of leading and lagging strands, respectively. Furthermore, GINS, a component of the replicative helicase complex that is composed of Sld5, Psf1, Psf2, and Psf3, was shown to interact weakly with all three replicative DNA Pols (α, δ, and ε) and to markedly stimulate the activities of Pol α and Pol ε. In vivo studies indicated that siRNA-targeted depletion of hPol δ and/or hPol ε reduced cell cycle progression and the rate of fork progression. Under the conditions used, we noted that depletion of Pol ε had a more pronounced inhibitory effect on cellular DNA replication than depletion of Pol δ. We suggest that reduction in the level of Pol δ may be less deleterious because of its collision-and-release role in lagging strand synthesis.

Oncogenic phosphatase Wip1 is a novel prognostic marker for lung adenocarcinoma patient survival.

DNA damage response pathways are important for maintaining genomic stability. The oncogenic phosphatase Wip1 plays a crucial role in DNA damage response by inhibiting several cell cycle proteins, including p53. Although Wip1 gene amplification has been reported in various primary tumors, including lung cancer, its biological significance for survival of primary lung tumor patients remains unclear. We investigated the expression of Wip1 in cancer epithelial cells immunohistochemically in 84 consecutive resected cases of lung adenocarcinoma. Increased Wip1 expression was observed in 54 (64.3%) of the 84 cases. Wip1 expression was found to be correlated significantly with two clinicopathological factors: γ-H2AX expression, and invasion to the pulmonary vein. A univariate analysis and log-rank test indicated a significant association between Wip1 expression and lower overall survival rate (P = 0.019 and P = 0.0099, respectively). A multivariate analysis also indicated a statistically significant association between increased Wip1 expression and lower overall survival rate (hazard ratio, 4.3; P = 0.026). The Ki67 index level was higher in the Wip1-positive group than in the negative group (P < 0.04, Mann-Whitney U-test). Moreover, in a subgroup analysis of only stage I patients, increased Wip1 expression was also significantly associated with a lower overall survival rate (P = 0.023, log-rank test). These results indicate that the increased expression of Wip1 in cancer epithelial cells has significant value for tumor progression and the clinical prognosis of patients with primary lung adenocarcinoma.

Nonsynonymous single nucleotide polymorphisms in DNA damage repair pathways and lung cancer risk.

BACKGROUND: Several reports have revealed the association between single nucleotide polymorphisms (SNPs) and the development of cancer. Although many SNPs have been investigated, they were tested individually. In this study, nonsynonymous SNPs present in DNA damage response genes were comprehensively analyzed for lung cancer susceptibility. METHODS: The authors selected 37 nonsynonymous SNPs in 23 genes involved in DNA damage repair pathways. Fifty lung adenocarcinoma patients resected at their institution between 2002 and 2005 and 50 individuals without any known history of cancer were recruited for a case-control study. RESULTS: Three variants (XRCC1 194Trp homozygotes, POLdelta1 119His homozygotes, and RAD9 239Arg heterozygotes) tended to coassociate with lung cancer risk. The authors analyzed and calculated whether the association between combinations of these 3 SNPs significantly affected the risk of lung cancer. Compared with carriers of either XRCC1 194Trp homozygote or RAD9 239Arg heterozygote variants, noncarriers were at a significantly decreased risk for lung cancer (odds ratio [OR], 0.282; confidence interval [CI], 0.089-0.893). The same results were found for the combination of POLdelta1 119His homozygotes and RAD9 239Arg heterozygotes (OR, 0.277; CI, 0.077-0.993). Moreover, compared with carriers that had at least 1 of the 3 variants, noncarriers showed a more significant decrease in risk (OR, 0.263; CI, 0.090-0.767). CONCLUSIONS: Analysis of the presence of XRCC1 194Trp homozygote, POLdelta1 119His homozygote, and RAD9 239Arg heterozygote variants revealed that their coassociation leads to a significant risk for the development of lung adenocarcinoma. Inclusive analyses of different SNPs were important in this cancer risk study.

Influence of the human cohesion establishment factor Ctf4/AND-1 on DNA replication.

Ctf4/AND-1 is a highly conserved gene product required for both DNA replication and the establishment of sister chromatid cohesion. In this report, we examined the mechanism of action of human Ctf4 (hCtf4) in DNA replication both in vitro and in vivo. Our findings show that the purified hCtf4 exists as a dimer and that the hCtf4 SepB domain likely plays a primary role determining the dimeric structure. hCtf4 binds preferentially to DNA template-primer structures, interacts directly with the replicative DNA polymerases (alpha, delta, and epsilon), and markedly stimulates the polymerase activities of DNA polymerases alpha and epsilon in vitro. Depletion of hCtf4 in HeLa cells by small interfering RNA resulted in G(1)/S phase arrest. DNA fiber analysis revealed that cells depleted of hCtf4 exhibited a rate of DNA replication slower than cells treated with control small interfering RNA. These findings suggest that in human cells, hCtf4 plays an essential role in DNA replication and its ability to stimulate the replicative DNA polymerases may contribute to this effect.

Phosphorylation of human DNA ligase I regulates its interaction with replication factor C and its participation in DNA replication and DNA repair.

Human DNA ligase I (hLigI) participates in DNA replication and excision repair via an interaction with proliferating cell nuclear antigen (PCNA), a DNA sliding clamp. In addition, hLigI interacts with and is inhibited by replication factor C (RFC), the clamp loader complex that loads PCNA onto DNA. Here we show that a mutant version of hLigI, which mimics the hyperphosphorylated M-phase form of hLigI, does not interact with and is not inhibited by RFC, demonstrating that inhibition of ligation is dependent upon the interaction between hLigI and RFC. To examine the biological relevance of hLigI phosphorylation, we isolated derivatives of the hLigI-deficient cell line 46BR.1G1 that stably express mutant versions of hLigI in which four serine residues phosphorylated in vivo were replaced with either alanine or aspartic acid. The cell lines expressing the phosphorylation site mutants of hLigI exhibited a dramatic reduction in proliferation and DNA synthesis and were also hypersensitive to DNA damage. The dominant-negative effects of the hLigI phosphomutants on replication and repair are due to the activation of cellular senescence, presumably because of DNA damage arising from replication abnormalities. Thus, appropriate phosphorylation of hLigI is critical for its participation in DNA replication and repair.

Cdc7-Drf1 kinase links chromosome cohesion to the initiation of DNA replication in Xenopus egg extracts.

To establish functional cohesion between replicated sister chromatids, cohesin is recruited to chromatin before S phase. Cohesin is loaded onto chromosomes in the G1 phase by the Scc2-Scc4 complex, but little is known about how Scc2-Scc4 itself is recruited to chromatin. Using Xenopus egg extracts as a vertebrate model system, we showed previously that the chromatin association of Scc2 and cohesin is dependent on the prior establishment of prereplication complexes (pre-RCs) at origins of replication. Here, we report that Scc2-Scc4 exists in a stable complex with the Cdc7-Drf1 protein kinase (DDK), which is known to bind pre-RCs and activate them for DNA replication. Immunodepletion of DDK from Xenopus egg extracts impairs chromatin association of Scc2-Scc4, a defect that is reversed by wild-type, but not catalytically inactive DDK. A complex of Scc4 and the N terminus of Scc2 is sufficient for chromatin loading of Scc2-Scc4, but not for cohesin recruitment. These results show that DDK is required to tether Scc2-Scc4 to pre-RCs, and they underscore the intimate link between early steps in DNA replication and cohesion.

Studies with the human cohesin establishment factor, ChlR1. Association of ChlR1 with Ctf18-RFC and Fen1.

Human ChlR1 (hChlR1), a member of the DEAD/DEAH subfamily of helicases, was shown to interact with components of the cohesin complex and play a role in sister chromatid cohesion. In order to study the biochemical and biological properties of hChlR1, we purified the protein from 293 cells and demonstrated that hChlR1 possesses DNA-dependent ATPase and helicase activities. This helicase translocates on single-stranded DNA in the 5' to 3' direction in the presence of ATP and, to a lesser extent, dATP. Its unwinding activity requires a 5'-singlestranded region for helicase loading, since flush-ended duplex structures do not support unwinding. The helicase activity of hChlR1 is capable of displacing duplex regions up to 100 bp, which can be extended to 500 bp by RPA or the cohesion establishment factor, the Ctf18-RFC (replication factor C) complex. We show that hChlR1 interacts with the hCtf18-RFC complex, human proliferating cell nuclear antigen, and hFen1. The interactions between Fen1 and hChlR1 stimulate the flap endonuclease activity of Fen1. Selective depletion of either hChlR1 or Fen1 by targeted small interfering RNA treatment results in the precocious separation of sister chromatids. These findings are consistent with a role of hChlR1 in the establishment of sister chromatid cohesion and suggest that its action may contribute to lagging strand processing events important in cohesion.

Crystal structure of the GINS complex and functional insights into its role in DNA replication.

The GINS complex, which contains the four subunits Sld5, Psf1, Psf2, and Psf3, is essential for both the initiation and progression of DNA replication in eukaryotes. GINS associates with the MCM2-7 complex and Cdc45 to activate the eukaryotic minichromosome maintenance helicase. It also appears to interact with and stimulate the polymerase activities of DNA polymerase epsilon and the DNA polymerase alpha-primase complex. To further understand the functional role of GINS, we determined the crystal structure of the full-length human GINS heterotetramer. Each of the four subunits has a major domain composed of an alpha-helical bundle-like structure. With the exception of Psf1, each of the other subunits has a small domain containing a three-stranded beta-sheet core. Each full-length protein in the crystal has unstructured regions that are all located on the surface of GINS and are probably involved in its interaction with other replication factors. The four subunits contact each other mainly through alpha-helices to form a ring-like tetramer with a central pore. This pore is partially plugged by a 16-residue peptide from the Psf3 N terminus, which is unique to some eukaryotic Psf3 proteins and is not required for tetramer formation. Removal of these N-terminal 16 residues of Psf3 from the GINS tetramer increases the opening of the pore by 80%, suggesting a mechanism by which accessibility to the pore may be regulated. The structural data presented here indicate that the GINS tetramer is a highly stable complex with multiple flexible surface regions.

A conserved physical and functional interaction between the cell cycle checkpoint clamp loader and DNA ligase I of eukaryotes.

DNA ligase I joins Okazaki fragments during DNA replication and completes certain excision repair pathways. The participation of DNA ligase I in these transactions is directed by physical and functional interactions with proliferating cell nuclear antigen, a DNA sliding clamp, and, replication factor C (RFC), the clamp loader. Here we show that DNA ligase I also interacts with the hRad17 subunit of the hRad17-RFC cell cycle checkpoint clamp loader, and with each of the subunits of its DNA sliding clamp, the heterotrimeric hRad9-hRad1-hHus1 complex. In contrast to the inhibitory effect of RFC, hRad17-RFC stimulates joining by DNA ligase I. Similar results were obtained with the homologous Saccharomyces cerevisiae proteins indicating that the interaction between the replicative DNA ligase and checkpoint clamp is conserved in eukaryotes. Notably, we show that hRad17 preferentially interacts with and specifically stimulates dephosphorylated DNA ligase I. Moreover, there is an increased association between DNA ligase I and hRad17 in S phase following DNA damage and replication blockage that occurs concomitantly with DNA damage-induced dephosphorylation of chromatin-associated DNA ligase I. Thus, our results suggest that the in vivo interaction between DNA ligase I and the checkpoint clamp loader is regulated by post-translational modification of DNA ligase I.

Human SHPRH is a ubiquitin ligase for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen.

Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI/SNF family of ATPases/helicases, and it possesses a C(3)HC(4) RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein, which, together with Mms2-Ubc13, promotes replication through DNA lesions via an error-free postreplicational repair pathway. Genetic evidence in yeast has indicated a role for Rad5 as a ubiquitin ligase in mediating the Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Here we show that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6-Rad18 and Mms2-Ubc13 complexes, and we show that SHPRH protein is a ubiquitin ligase indispensable for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raises the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans.

His239Arg SNP of HRAD9 is associated with lung adenocarcinoma.

BACKGROUND: It was previously reported that a functional human (h) Rad9 protein accumulated in the nuclei of non-small cell lung carcinoma (NSCLC) cells. Those experiments, however, did not examine whether the hRad9 gene was mutated in those cells. The sequence of the HRAD9 gene in NSCLC cells was investigated. METHODS: The sequence of the HRAD9 was examined in tumor and peripheral normal lung tissues obtained from 50 lung adenocarcinoma patients during surgery. The expression of its mRNA using reverse transcription polymerase chain reaction (RT-PCR) was also examined. RESULTS: No sequence alterations were detected in the HRAD9 gene, which was found to be normally transcribed in surgically resected lung carcinoma cells. However, in eight (16.0%) cases a single nucleotide polymorphism (SNP) was observed at the second position of codon 239 (His/Arg heterozygous variant) of the gene. This frequency was significantly higher than that found in the normal population. CONCLUSIONS: Whereas the capacity to produce a functional hRad9 protein was intact in lung adenocarcinoma cells, a nonsynonymous SNP of HRAD9 was detected that might be associated with the development of lung adenocarcinoma.

Proximity-induced activation of human Cdc34 through heterologous dimerization.

Cdc34 is an E2-conjugating enzyme required for catalyzing the polyubiquitination reaction mediated by the Skp1.CUL1.F-box (SCF) protein E3 ubiquitin (Ub) ligase. Here, we show that the activity of human Cdc34 in the Ub-Ub ligation reaction was enhanced dramatically by SCF's core Ub ligase module, composed of a heterodimeric complex formed by the ROC1 RING finger protein and the CUL1 C terminus that contains a Nedd8 moiety covalently conjugated at K720. Unexpectedly, we found that N-terminal fusion of a GST moiety to human Cdc34 generated dimeric GST-Cdc34 that was constitutively active in supporting the assembly of K48-linked polyUb chains independently of SCF. Furthermore, fusion of a FK506-binding protein (FKBP) to the N terminus of human Cdc34 yielded FKBP-Cdc34 that was induced to form a dimer upon treatment with the chemical inducer AP20187. The AP20187-induced dimeric form of FKBP-Cdc34 was substantially more active than the monomer in catalyzing Ub-Ub ligation. Thus, juxtaposition of human Cdc34 activates its catalytic capability, suggesting that the SCF-mediated polyubiquitination reaction may require the conversion of Cdc34 from an inactive monomer to a highly active dimeric form.

Accumulation of hRad9 protein in the nuclei of nonsmall cell lung carcinoma cells.

BACKGROUND: DNA damage sensor proteins have received much attention as upstream components of the DNA damage checkpoint signaling pathway that are required for cell cycle control and the induction of apoptosis. Deficiencies in these proteins are directly linked to the accumulation of gene mutations, which can induce cellular transformation and result in malignant disease. METHODS: Using 48 sets of tumor tissue specimens and peripheral normal lung tissue specimens from 48 patients with nonsmall cell lung carcinoma (NSCLC) who underwent surgery, the authors investigated the expression of hRad9 protein, a member of the human DNA damage sensor family, using immunohistochemical and Western blot analyses. RESULTS: Immunohistochemical analysis detected the accumulation of hRad9 in the nuclei of tumor cells in 16 tumor tissue specimens, (33% of tumor tissue specimens examined). Western blot analysis also revealed elevated levels of phosphorylated hRad9 protein in NSCLC cells that was accompanied by the detection of phosphorylated Chk1, a protein kinase that regulates the downstream signaling of the DNA damage checkpoint pathway. Furthermore, strong expression of hRad9 was correlated with an increase in Ki-67 expression index in the tumor cells that were examined. CONCLUSIONS: The findings made in the current study suggest that Rad9 expression may play an important role in cell cycle control in NSCLC cells and may influence NSCLC cell phenotype.

Atomic force microscopic analysis of the binding of the Schizosaccharomyces pombe origin recognition complex and the spOrc4 protein with origin DNA.

In eukaryotes, the initiation of DNA replication requires the interaction between origin sequences and the origin recognition complex (ORC), which is highly conserved. In this report, atomic force microscopy (AFM) was used to examine the binding of Schizosaccharomyces pombe (sp) ORC and the spOrc4 protein with the sp autonomously replicating sequence 1 (ars1). AFM imaging revealed that spORC binding to ars1 occurred solely through spOrc4p and depended on the N-terminal AT-hook domains present in spOrc4p. At high molar ratios of spORC (or spOrc4p alone) to DNA (6:1), all of the input ars1 was bound in a one protein complex to one plasmid manner. Restriction digestion and AFM analysis of protein-DNA fragments revealed the presence of two binding sites in ars1. One site mapped to a region centered at nucleotide 838 of ars1 previously detected by DNase I protection that was reported to be essential for the autonomously replicating sequence activity of ars1. The second site mapped to a previously uncharacterized region centered at nucleotide 1148. AFM showed that the length of the DNA fragment complexed with either spORC or spOrc4p was shortened by approximately 140 bp, suggesting the wrapping of two turns of the DNA around the spOrc4p alone as well as the spOrc4p in spORC. We also show that treatment of the spORC (spOrc4p)-ars1 complex with topoisomerase I induced a negative shift in the topoisomer distribution. These findings suggest that the binding of spORC to origin DNA alters the structure of the DNA. Thus, in the case of spORC, due to its unusual spOrc4p, at least two factors are likely to influence ars1 activation. These include the selective binding of the complex to A- and T-rich regions and the alteration of the DNA structure due to its wrapping around spOrc4p.