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C F Dutta

Publications and source records attributed to C F Dutta.

6 recordsLinked to original sources
PubMed2003

Characterization of an EcoR124I restriction-modification enzyme produced from a deleted form of the DNA-binding subunit, which results in a novel DNA specificity.

We purified and characterized both the methyltransferase and the endonuclease containing the HsdS delta 50 subunit (type I restriction endonucleases are composed of three subunits--HsdR required for restriction, HsdM required for methylation and HsdS responsible for DNA recognition) produced from the deletion mutation hsdS delta 50 of the type IC R-M system EcoR 124I; this mutant subunit lacks the C-terminal 163 residues of HsdS and produces a novel DNA specificity. Analysis of the purified HsDs delta 50 subunit indicated that during purification it is subject to partial proteolysis resulting in removal of approximately 1 kDa of the polypeptide at the C-terminus. This proteolysis prevented the purification of further deletion mutants, which were determined as having a novel DNA specificity in vivo. After biochemical characterization of the mutant DNA methyltransferase (MTase) and restriction endonuclease we found only one difference comparing with the wild-type enzyme--a significantly higher binding affinity of the MTase for the two substrates of hemimethylated and fully methylated DNA. This indicates that MTase delta 50 is less able to discriminate the methylation status of the DNA during its binding. However, the mutant MTase still preferred hemimethylated DNA as the substrate for methylation. We fused the hsdM and hsdS delta 50 genes and showed that the HsdM-HsdS delta 50 fusion protein is capable of dimerization confirming the model for assembly of this deletion mutant.

Amino Acid Sequence↗
PubMed2000

A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway.

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M(2)S(1). Here, we analysed the impact on the restriction and modification activities of the change Trp(212)-->Arg in the distal border of the central conserved region of the EcoR124I HsdS subunit. We demonstrate that this point mutation significantly influences the ability of the mutant HsdS subunit to assemble with the HsdM subunit to produce a functional MTase. As a consequence of this, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, HsdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentrations in vitro of HsdR, restoring restriction activity.

Catalysis↗
PubMed2000

Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties.

Two temperature-sensitive mutations in the hsdS gene, which encodes the DNA specificity subunit of the type IA restriction-modification system EcoKI, designated Sts1 (Ser(340)Phe) and Sts2 (Ala(204)Thr) had a different impact on restriction-modification functions in vitro and in vivo. The enzyme activities of the Sts1 mutant were temperature-sensitive in vitro and were reduced even at 30 degrees C (permissive temperature). Gel retardation assays revealed that the Sts1 mutant had significantly decreased DNA binding, which was temperature-sensitive. In contrast the Sts2 mutant did not show differences from the wild-type enzyme even at 42 degrees C. Unlike the HsdSts1 subunit, the HsdSts2 subunit was not able to compete with the wild-type subunit in assembly of the restriction enzyme in vivo, suggesting that the Sts2 mutation affects subunit assembly. Thus, it appears that these two mutations map two important regions in HsdS subunit responsible for DNA-protein and protein-protein interactions, respectively.

Adenosine Triphosphatases↗
PubMed1999

A highly conserved plasmid from the extreme thermophile Thermotoga maritima MC24 is a member of a family of plasmids distributed worldwide.

We have screened Thermotoga strains, isolated from hydrothermal vents near the Kuril Islands, for the presence of plasmid DNA. The miniplasmid pMC24 was isolated from the extreme thermophilic eubacteria Thermotoga maritima and sequenced, showing it to be a plasmid of 846 bp. It was found, from a search of the databases, to be closely related to the previously described Thermotoga miniplasmid pRQ7, isolated from a strain found on the Azore Islands, and was distinguished by only two point mutations. These changes resulted in two consecutive frameshifts altering a region encoding 9 amino acids in the Rep-coding region. We have also shown that pMC24, as with pRQ7, is negatively supercoiled. It seems that negatively supercoiled miniplasmids related to pRQ7 are spread worldwide and strongly maintained among Thermotoga strains.

Base Sequence↗
PubMed1992

High-level expression of the cloned genes encoding the subunits of and intact DNA methyltransferase, M.EcoR124.

We have cloned the genes coding for the two subunits (HsdM and HsdS) of the type-I DNA methyltransferase (MTase), M.EcoR124, into the specially constructed expression vector, pJ119. These subunits have been synthesized together as an intact MTase. We have also cloned the individual subunit-encoding genes under the control of the T7 gene 10 promoter or the lacUV5 promoter. High levels of expression have been obtained in all cases. While HsdM was found to be soluble, HsdS was insoluble. However, in the presence of the co-produced HsdM subunit, HsdS was found in the soluble fraction as part of an active MTase. We have partially purified the cloned multi-subunit enzyme and shown that it is capable of DNA methylation both in vivo and in vitro.

Amino Acid Sequence↗