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RNase HI (rnase + hi)

Distribution by Scientific Domains

Life Sciences	40%

Selected Abstracts

Destabilization of psychrotrophic RNase HI in a localized fashion as revealed by mutational and X-ray crystallographic analyses

FEBS JOURNAL, Issue 2 2009
Muhammad S. Rohman
The Arg97 , Gly and Asp136 , His mutations stabilized So-RNase HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 by 5.4 and 9.7 °C, respectively, in Tm, and 3.5 and 6.1 kJ·mol,1, respectively, in ,G(H2O). These mutations also stabilized the So-RNase HI derivative (4×-RNase HI) with quadruple thermostabilizing mutations in an additive manner. As a result, the resultant sextuple mutant protein (6×-RNase HI) was more stable than the wild-type protein by 28.8 °C in Tm and 27.0 kJ·mol,1 in ,G(H2O). To analyse the effects of the mutations on the protein structure, the crystal structure of the 6×-RNase HI protein was determined at 2.5 Å resolution. The main chain fold and interactions of the side-chains of the 6×-RNase HI protein were basically identical to those of the wild-type protein, except for the mutation sites. These results indicate that all six mutations independently affect the protein structure, and are consistent with the fact that the thermostabilizing effects of the mutations are roughly additive. The introduction of favourable interactions and the elimination of unfavourable interactions by the mutations contribute to the stabilization of the 6×-RNase HI protein. We propose that So-RNase HI is destabilized when compared with its mesophilic and thermophilic counterparts in a localized fashion by increasing the number of amino acid residues unfavourable for protein stability. [source]

Identification of RNase HII from psychrotrophic bacterium, Shewanella sp.

FEBS JOURNAL, Issue 10 2006
SIB1 as a high-activity type RNase H
The gene encoding RNase HII from the psychrotrophic bacterium, Shewanella sp. SIB1 was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RNase HII is a monomeric protein with 212 amino acid residues and shows an amino acid sequence identity of 64% to E. coli RNase HII. The enzymatic properties of SIB1 RNase HII, such as metal ion preference, pH optimum, and cleavage mode of substrate, were similar to those of E. coli RNase HII. SIB1 RNase HII was less stable than E. coli RNase HII, but the difference was marginal. The half-lives of SIB1 and E. coli RNases HII at 30 °C were ,,30 and 45 min, respectively. The midpoint of the urea denaturation curve and optimum temperature of SIB1 RNase HII were lower than those of E. coli RNase HII by ,,0.2 m and ,,5 °C, respectively. However, SIB1 RNase HII was much more active than E. coli RNase HII at all temperatures studied. The specific activity of SIB1 RNase HII at 30 °C was 20 times that of E. coli RNase HII. Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII represent low- and high-activity type RNases H, respectively, in SIB1. In contrast, RNases HI and HII represent high- and low-activity type RNases H, respectively, in E. coli. We propose that bacterial cells usually contain low- and high-activity type RNases H, but these types are not correlated with RNase H families. [source]

The absence of ribonuclease H1 or H2 alters the sensitivity of Saccharomyces cerevisiae to hydroxyurea, caffeine and ethyl methanesulphonate: implications for roles of RNases H in DNA replication and repair

GENES TO CELLS, Issue 10 2000
Arulvathani Arudchandran
Background RNA of RNA-DNA hybrids can be degraded by ribonucleases H present in all organisms including the eukaryote Saccharomyces cerevisiae. Determination of the number and roles of the RNases H in eukaryotes is quite feasible in S. cerevisiae. Results Two S. cerevisiae RNases H, related to Escherichia coli RNase HI and HII, are not required for growth under normal conditions, yet, compared with wild-type cells, a double-deletion strain has an increased sensitivity to hydroxyurea (HU) and is hypersensitive to caffeine and ethyl methanesulphonate (EMS). In the absence of RNase H1, RNase H2 activity increases, and cells are sensitive to EMS but not HU and are more tolerant of caffeine; the latter requires RNase H2 activity. Cells missing only RNase H2 exhibit increased sensitive to HU and EMS but not caffeine Conclusions Mutant phenotypes infer that some RNA-DNA hybrids are recognized by both RNases H1 and H2, while other hybrids appear to be recognized only by RNase H2. Undegraded RNA-DNA hybrids have an effect when DNA synthesis is impaired, DNA damage occurs or the cell cycle is perturbed by exposure to caffeine suggesting a role in DNA replication/repair that can be either beneficial or detrimental to cell viability. [source]

RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli

MOLECULAR MICROBIOLOGY, Issue 1 2004
Imad Baaklini
Summary It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow. Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants. However, how excess negative supercoiling inhibits growth is still unknown. We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R-loop, can partially compensate for the growth defects because of the absence of topoisomerase I. In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells. We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced. Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full-length mRNAs when RNase HI is not overproduced. Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way. All together, our results suggest that excess negative supercoiling promotes the formation of R-loops, which, in turn, inhibit RNA synthesis. [source]