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K R Sreekumar

Publications and source records attributed to K R Sreekumar.

9 recordsLinked to original sources
PubMed2001

Computational analysis of human disease-associated genes and their protein products.

The complete genome sequences for human, Drosophila melanogaster and Arabidopsis thaliana have been reported recently. With the availability of complete sequences for many bacteria and archaea, and five eukaryotes, comparative genomics and sequence analysis are enabling us to identify counterparts of many human disease genes in model organisms, which in turn should accelerate the pace of research and drug development to combat human diseases. Continuous improvement of specialized protein databases, together with sensitive computational tools, have enhanced the power and reliability of computational prediction of protein function.

Animals↗
PubMed2000

The simian virus 40 core origin contains two separate sequence modules that support T-antigen double-hexamer assembly.

Using subfragments of the simian virus 40 (SV40) core origin, we demonstrate that two alternative modules exist for the assembly of T-antigen (T-ag) double hexamers. Pentanucleotides 1 and 3 and the early palindrome (EP) constitute one assembly unit, while pentanucleotides 2 and 4 and the AT-rich region constitute a second, relatively weak, assembly unit. Related studies indicate that on the unit made up of pentanucleotide 1 and 3 and the EP assembly unit, the first hexamer forms on pentanucleotide 1 and that owing to additional protein-DNA and protein-protein interactions, the second hexamer is able to form on pentanucleotide 3. Oligomerization on the unit made up of pentanucleotide 2 and 4 and the AT-rich region is initiated by assembly of a hexamer on pentanucleotide 4; subsequent formation of the second hexamer takes place on pentanucleotide 2. Given that oligomerization on the SV40 origin is limited to double-hexamer formation, it is likely that only a single module is used for the initial assembly of T-ag double hexamers. Finally, we discuss the evidence that nucleotide hydrolysis is required for the remodeling events that result in the utilization of the second assembly unit.

Adenosine Triphosphate↗
PubMed2000

Phosphorylation of simian virus 40 T antigen on Thr 124 selectively promotes double-hexamer formation on subfragments of the viral core origin.

Cell cycle-dependent phosphorylation of simian virus 40 (SV40) large tumor antigen (T-ag) on threonine 124 is essential for the initiation of viral DNA replication. A T-ag molecule containing a Thr-->Ala substitution at this position (T124A) was previously shown to bind to the SV40 core origin but to be defective in DNA unwinding and initiation of DNA replication. However, exactly what step in the initiation process is defective as a result of the T124A mutation has not been established. Therefore, to better understand the control of SV40 replication, we have reinvestigated the assembly of T124A molecules on the SV40 origin. Herein it is demonstrated that hexamer formation is unaffected by the phosphorylation state of Thr 124. In contrast, T124A molecules are defective in double-hexamer assembly on subfragments of the core origin containing single assembly units. We also report that T124A molecules are inhibitors of T-ag double hexamer formation. These and related studies indicate that phosphorylation of T-ag on Thr 124 is a necessary step for completing the assembly of functional double hexamers on the SV40 origin. The implications of these studies for the cell cycle control of SV40 DNA replication are discussed.

Amino Acid Substitution↗
PubMed1999

Effect of cold shock on lipid A biosynthesis in Escherichia coli. Induction At 12 degrees C of an acyltransferase specific for palmitoleoyl-acyl carrier protein.

Palmitoleate is not present in lipid A isolated from Escherichia coli grown at 30 degrees C or higher, but it comprises approximately 11% of the fatty acyl chains of lipid A in cells grown at 12 degrees C. The appearance of palmitoleate at 12 degrees C is accompanied by a decline in laurate from approximately 18% to approximately 5.5%. We now report that wild-type E. coli shifted from 30 degrees C to 12 degrees C acquire a novel palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase that acts on the key lipid A precursor Kdo2-lipid IVA. The palmitoleoyl transferase is induced more than 30-fold upon cold shock, as judged by assaying extracts of cells shifted to 12 degrees C. The induced activity is maximal after 2 h of cold shock, and then gradually declines but does not disappear. Strains harboring an insertion mutation in the lpxL(htrB) gene, which encodes the enzyme that normally transfers laurate from lauroyl-ACP to Kdo2-lipid IVA (Clementz, T., Bednarski, J. J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095-12102) are not defective in the cold-induced palmitoleoyl transferase. Recently, a gene displaying 54% identity and 73% similarity at the protein level to lpxL was found in the genome of E. coli. This lpxL homologue, designated lpxP, encodes the cold shock-induced palmitoleoyl transferase. Extracts of cells containing lpxP on the multicopy plasmid pSK57 exhibit a 10-fold increase in the specific activity of the cold-induced palmitoleoyl transferase compared with cells lacking the plasmid. The elevated specific activity of the palmitoleoyl transferase under conditions of cold shock is attributed to greatly increased levels of lpxP mRNA. The replacement of laurate with palmitoleate in lipid A may reflect the desirability of maintaining the optimal outer membrane fluidity at 12 degrees C.

Acyl Carrier Protein↗
PubMed1999

Sequence requirements for the assembly of simian virus 40 T antigen and the T-antigen origin binding domain on the viral core origin of replication.

The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd(131-260)) have been determined. Binding of the purified T-ag-obd(131-260) is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers.

Animals↗
PubMed1998

Assembly of T-antigen double hexamers on the simian virus 40 core origin requires only a subset of the available binding sites.

Initiation of simian virus 40 (SV40) DNA replication is dependent upon the assembly of two T-antigen (T-ag) hexamers on the SV40 core origin. To further define the oligomerization mechanism, the pentanucleotide requirements for T-ag assembly were investigated. Here, we demonstrate that individual pentanucleotides support hexamer formation, while particular pairs of pentanucleotides suffice for the assembly of T-ag double hexamers. Related studies demonstrate that T-ag double hexamers formed on "active pairs" of pentanucleotides catalyze a set of previously described structural distortions within the core origin. For the four-pentanucleotide-containing wild-type SV40 core origin, footprinting experiments indicate that T-ag double hexamers prefer to bind to pentanucleotides 1 and 3. Collectively, these experiments demonstrate that only two of the four pentanucleotides in the core origin are necessary for T-ag assembly and the induction of structural changes in the core origin. Since all four pentanucleotides in the wild-type origin are necessary for extensive DNA unwinding, we concluded that the second pair of pentanucleotides is required at a step subsequent to the initial assembly process.

Antigens, Viral, Tumor↗
PubMed1997

Initiation of SV40 DNA replication in vitro: analysis of the role played by sequences flanking the core origin on initial synthesis events.

Replication initiation events are suppressed over the SV40 core origin in vitro; they are also greatly reduced over sequences flanking the origin which contain binding sites for several transcription factors. To address the biochemical basis for the gap in initiation events over the flanking sequences, initial synthesis events have been characterized on templates lacking these sequences. Herein, it is demonstrated that previously functional initiation sites are nearly inactive when moved to positions that are proximal to the core origin. Thus, the gap in initiation events depends, in part, on the proximity of the initiation sites to the SV40 core origin. Additional experiments demonstrate that removal of the flanking sequences had little or no effect on DNA unwinding or on the efficiency of initiation of DNA synthesis in vitro. These results indicate that, under our in vitro conditions, initiation of SV40 DNA synthesis is not enhanced by binding of transcription factors to the flanking sequences.

Antigens, Viral, Tumor↗
PubMed1997

Purification of the simian virus 40 (SV40) T antigen DNA-binding domain and characterization of its interactions with the SV40 origin.

To better define protein-DNA interactions at a eukaryotic origin, the domain of simian virus 40 (SV40) large T antigen that specifically interacts with the SV40 origin has been purified and its binding to DNA has been characterized. Evidence is presented that the affinity of the purified T antigen DNA-binding domain for the SV40 origin is comparable to that of the full-length T antigen. Furthermore, stable binding of the T antigen DNA-binding domain to the SV40 origin requires pairs of pentanucleotide recognition sites separated by approximately one turn of a DNA double helix and positioned in a head-to-head orientation. Although two pairs of pentanucleotides are present in the SV40 origin, footprinting and band shift experiments indicate that binding is limited to dimer formation on a single pair of pentanucleotides. Finally, it is demonstrated that the T antigen DNA-binding domain interacts poorly with single-stranded DNA.

Antigens, Polyomavirus Transforming↗