Biomedical subjects

Vladimir P Bermudez

Publications and source records attributed to Vladimir P Bermudez.

8 recordsLinked to original sources

PubMed2006

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.

Adenocarcinoma↗

PubMed2005

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.

Adult↗

PubMed2004

A conserved interaction between the replicative clamp loader and DNA ligase in eukaryotes: implications for Okazaki fragment joining.

The recruitment of DNA ligase I to replication foci and the efficient joining of Okazaki fragments is dependent on the interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA). Although the PCNA sliding clamp tethers DNA ligase I to nicked duplex DNA circles, the interaction does not enhance DNA joining. This suggests that other factors may be involved in the joining of Okazaki fragments. In this study, we describe an association between replication factor C (RFC), the clamp loader, and DNA ligase I in human cell extracts. Subsequently, we demonstrate that there is a direct physical interaction between these proteins that involves both the N- and C-terminal domains of DNA ligase I, the N terminus of the large RFC subunit p140, and the p36 and p38 subunits of RFC. Although RFC inhibited DNA joining by DNA ligase I, the addition of PCNA alleviated inhibition by RFC. Notably, the effect of PCNA on ligation was dependent on the PCNA-binding site of DNA ligase I. Together, these results provide a molecular explanation for the key in vivo role of the DNA ligase I/PCNA interaction and suggest that the joining of Okazaki fragments is coordinated by pairwise interactions among RFC, PCNA, and DNA ligase I.

Binding Sites↗

PubMed2003

The alternative Ctf18-Dcc1-Ctf8-replication factor C complex required for sister chromatid cohesion loads proliferating cell nuclear antigen onto DNA.

The linkage of sister chromatids after DNA replication ensures the faithful inheritance of chromosomes by daughter cells. In budding yeast, the establishment of sister chromatid cohesion requires Ctf8, Dcc1, and Ctf18, a homologue of the p140 subunit of the replication factor C (RFC). In this report we demonstrate that in 293T cells, Flag-tagged Ctf18 forms a seven-subunit cohesion-RFC complex comprised of Ctf18, Dcc1, Ctf8, RFCp40, RFCp38, RFCp37, and RFCp36 (Ctf18-RFC). We demonstrate that a stoichiometric heteroheptameric Ctf18-RFC complex can be assembled by coexpressing the seven proteins in baculovirus-infected insect cells. In addition, the two other stable subcomplexes were formed, which include a pentameric complex comprised of Ctf18, RFCp40, RFCp38, RFCp37, and RFCp36 and a dimeric Dcc1-Ctf8. Both the five- and seven-subunit Ctf18-RFC complexes bind to single-stranded and primed DNAs and possess weak ATPase activity that is stimulated by the addition of primed DNA and proliferating cell nuclear antigen (PCNA). These complexes catalyzed the ATP-dependent loading of PCNA onto primed and gapped DNA but not onto double-stranded nicked or single-stranded circular DNAs. Consistent with these observations, both Ctf18-RFC complexes substituted for the replicative RFC in the PCNA-dependent DNA polymerase delta-catalyzed DNA replication reaction. These results support a model in which sister chromatid cohesion is linked to DNA replication.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2003

Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro.

The human DNA damage sensors, Rad17-replication factor C (Rad17-RFC) and the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, are thought to be involved in the early steps of the DNA damage checkpoint response. Rad17-RFC and the 9-1-1 complex have been shown to be structurally similar to the replication factors, RFC clamp loader and proliferating cell nuclear antigen polymerase clamp, respectively. Here, we demonstrate functional similarities between the replication and checkpoint clamp loader/DNA clamp pairs. When all eight subunits of the two checkpoint complexes are coexpressed in insect cells, a stable Rad17-RFC/9-1-1 checkpoint supercomplex forms in vivo and is readily purified. The two individually purified checkpoint complexes also form a supercomplex in vitro, which depends on ATP and is mediated by interactions between Rad17 and Rad9. Rad17-RFC binds to nicked circular, gapped, and primed DNA and recruits the 9-1-1 complex in an ATP-dependent manner. Electron microscopic analyses of the reaction products indicate that the 9-1-1 ring is clamped around the DNA.

Adenosine Triphosphate↗

PubMed2002

The influence of the Cdc27 subunit on the properties of the Schizosaccharomyces pombe DNA polymerase delta.

Schizosaccharomyces pombe DNA polymerase (pol) delta contains four subunits, pol 3, Cdc1, Cdc27, and Cdm1. In this report, we examined the role of Cdc27 on the structure and activity of pol delta. We show that the four-subunit complex is monomeric in structure, in contrast to the previous report that it was a dimer (Zuo, S., Bermudez, V., Zhang, G., Kelman, Z., and Hurwitz, J. (2000) J. Biol. Chem. 275, 5153-5162). This discrepancy between the earlier and recent observations was traced to the marked asymmetric shape of Cdc27. Cdc27 contains two critical domains that govern its role in activating pol delta. The N-terminal region (amino acids (aa) 1-160) binds to Cdc1 and its extreme C-terminal end (aa 362-369) interacts with proliferating cell nuclear antigen (PCNA). Mutants of S. pombe pol delta, containing truncated Cdc27 derivatives deficient in binding to PCNA, supported DNA replication less processively than the wild-type complex. Fusion of a minimal PCNA-binding motif (aa 352-372) to C-terminally truncated Cdc27 derivatives restored processive DNA synthesis in vitro. In vivo, the introduction of these fused Cdc27 derivatives into cdc27Delta cells conferred viability. These data support the model in which Cdc27 plays an essential role in DNA replication by recruiting PCNA to the pol delta holoenzyme.

Amino Acid Motifs↗

PubMed2002

A Mammalian bromodomain protein, brd4, interacts with replication factor C and inhibits progression to S phase.

Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G(1) upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G(1) to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.

3T3 Cells↗

PubMed2002

Stimulation of DNA replication from the polyomavirus origin by PCAF and GCN5 acetyltransferases: acetylation of large T antigen.

The PCAF and GCN5 acetyltransferases, but not p300 or CBP, stimulate DNA replication when tethered near the polyomavirus origin. Replication stimulation by PCAF and GCN5 is blocked by mutational inactivation of their acetyltransferase domains but not by deletion of sequences that bind p300 or CBP. Acetylation of histones near the polyomavirus origin assembled into chromatin in vivo is not detectably altered by expression of these acetyltransferases. PCAF and GCN5 interact with polyomavirus large T antigen in vivo, PCAF acetylates large T antigen in vitro, and large T-antigen acetylation in vivo is dependent upon the integrity of the PCAF acetyltransferase domain. These data suggest replication stimulation occurs through recruitment of large T antigen to the origin and acetylation by PCAF or GCN5.

3T3 Cells↗