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Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The rnh gene of Escherichia coli encodes RNase H. rnh mutants display at least two phenotypes: (1) they require functional RecBCD enzyme for growth; thus rnh-339::cat recB270 (Ts) and rnh-339::cat recC271 (Ts) strains are temperature sensitive for growth; (2) rnh mutants permit replication that is independent of the chromosomal origin, presumably by failing to remove RNA-DNA hybrids from which extra-original replication can be primed. We report here that manifestation of these two phenotypes occurs at different levels of RNase H function; we have examined partially functional rnh mutants for their in vitro RNase H activity, their ability to rescue viability in recB or recC cells and their ability to permit growth of mutants incapable of using oriC [dnaA (Ts)].
Mol Gen Genet 1991 Jul
PMID:Correlation of activity with phenotypes of Escherichia coli partial function mutants of rnh, the gene encoding RNase H. 165 Sep 9

We have cloned genes encoding RNase H from Escherichia coli rnh mutants, Salmonella typhimurium and Saccharomyces cerevisiae. Selection was accomplished by suppression of the temperature-sensitive growth phenotype of Escherichia coli strains containing the rnh-339::cat and either recB270 (Ts) or recC271 (Ts) mutations. RNases H from E. coli and S. typhimurium contained 155 amino acid residues and differed at only 11 positions. The S. cerevisiae and E. coli RNases H were about 30% homologous. A comparison of the amino acid sequences of several RNases H from cellular and retroviral sources revealed some strongly conserved regions as well as variable regions; the carboxyl-terminus was particularly variable. The overlapping, divergent promoter gene organization found in E. coli was observed to be present in S. typhimurium.
Mol Gen Genet 1991 Jul
PMID:Selective cloning of genes encoding RNase H from Salmonella typhimurium, Saccharomyces cerevisiae and Escherichia coli rnh mutant. 165 Sep 10

The C-terminal region of human immunodeficiency virus (HIV) reverse transcriptase (RT) contains the domain responsible for RNase H activity. To determine the importance of this RNase H domain, specific changes in the C-terminal region of a recombinant RT expressed in Escherichia coli were introduced by amino acid substitutions and specific deletions. The enzyme activities of purified wild-type and mutant RT/RNase H proteins, standardized for protein content, were compared by filter assays and thermal inactivation kinetics. A point mutation of His 539----Asn produced an enzyme with a marked thermolabile RNase H function (nine-fold increase in inactivation), whereas RT function was only marginally more labile than that of the wild-type (two-fold). A second mutation, His 539----Asp, impaired both enzyme activities to a similar degree (four- to five-fold). A C-terminal deletion of 19 amino acids (aa) (aa 540 to 558) and a C-terminal truncation of 21 aa (aa 540 to 560) reduced RT as well as RNase H activity. A 130 aa deletion enzyme exhibited no RNase H activity and insufficient RT activity to allow inactivation studies. Two mutants, the 19 aa deletion and His----Asn, were introduced into proviral HIV-1 DNA clones to determine whether changes in enzyme activity, particularly RNase H activity, affected virus infectivity. Both mutants were non-infectious, indicating that the C-terminal 19 to 21 amino acids and His 539 of the RT/RNase H protein are essential for HIV replication. These results are consistent with the assumption that RNase H is essential for the infectivity of HIV-1.
J Gen Virol 1991 Jan
PMID:Mutations within the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase abolish virus infectivity. 170 63

Destabilizing events required for subsequent cotranslational disassembly of tobacco mosaic virus (TMV) particles in vitro were studied. Brief treatment of U-32P-labelled TMV (strain vulgare or U2) with 1% SDS exposed only 2.5% of the RNA (160 5' nucleotides) in a susceptible subpopulation of virions. Limited uncoating occurred almost immediately and appeared to be synchronous because the amount of 5' oligonucleotide marker (omega) recovered remained constant throughout a 15 min period in SDS. Additional RNase T1-sensitive oligonucleotides were exposed only after 1 to 2 min in SDS. Coat protein (CP) subunits released from virions 'destabilized' by ultracentrifugation at between pH 7.2 and 9.2 were quantified using L-[35S]methionine-labelled particles of TMV strain U2. CP recovery and virus particle translation results were consistent with increasing numbers of virions uncoating for approximately 200 nucleotides. In the presence of sparsomycin (SPN), the TMV strain vulgare 5' leader and the first AUG codon can bind two 80S ribosomes. Electron microscopy of pH 7.5-treated TMV particles incubated in SPN-treated wheatgerm extract or rabbit reticulocyte lysate, showed that approximately 10% of virions complexed with one ribosome and approximately 10% with two bound ribosomes, confirming that omega at least had been uncoated. Nucleocapsids in these complexes were shorter than untreated TMV by 9 to 10 nm (i.e. equivalent to 192 to 217 nucleotides exposed). The template activities of virions pretreated at pH 7.2 to 9.2 were destroyed by RNase H when short cDNAs were hybridized to sequences at, or immediately 3' to, the first AUG codon. We propose that the complete 5' leader of TMV RNA interacts weakly with CP subunits and that this micro-instability is due to the absence of G residues and is essential for initiation of cotranslational virus disassembly.
J Gen Virol 1991 Apr
PMID:Complete uncoating of the 5' leader sequence of tobacco mosaic virus RNA occurs rapidly and is required to initiate cotranslational virus disassembly in vitro. 184 66

Two modes of ColE1 DNA replication are known, one dependent on RNase H, and the other RNase H independent. The cer114 mutant of the ColE1 replicon is defective in both modes and carries a single base pair alteration 95 bp upstream of the replication origin. An Escherichia coli mutant which restored maintenance of the cer114 replicon was isolated. This host suppressor mutant is defective in RNase H and carries a herC mutation located at 62 min of the E. coli chromosome. The herC mutation is recessive to its wild-type allele and supports maintenance of the mutant replicon in the absence of RNase H. The herC mutation alone conferred cold-sensitive growth, suggesting that the herC gene product is essential for cell growth. The 1832 bp E. coli DNA fragment, containing the wild-type allele of the herC mutation, was cloned and an open reading frame for the HerC protein was determined.
Mol Gen Genet 1989 Nov
PMID:Isolation and characterization of herC, a mutation of Escherichia coli affecting maintenance of ColE1. 256 Jan 34

Escherichia coli rnh mutants deficient in ribonuclease H (RNase H) are capable of DNA replication in the absence of protein synthesis. This constitutive stable DNA replication (SDR) is dependent upon the recA+ gene product. The requirement of SDR for recA+ can be suppressed by rin mutations (for recA+-independent), or by lexA(Def) mutations which inactivate the LexA repressor. Thus, there are at least three genetically distinct types of SDR in rnh mutants: recA+-dependent SDR seen in rnh- rin+ lexA+ strains, recA+-independent in rnh- rin- lexA+, and recA+-independent in rnh- rin+ lexA(Def). The expression of SDR in rin- and lexA(Def) mutants demonstrated a requirement for RNA synthesis and for the absence of RNase H. The suppression of the recA+ requirement by rin mutations was shown to depend on some new function of the recF+ gene product. In contrast, the suppression by lexA-(Def) mutations was not dependent on recF+. The lexA3 mutation inhibited recA+-dependent SDR via reducing the amount of recA+ activity available, and was suppressed by the recAo254 mutation. The SDR in rnh- rin- cells was also inhibited by the lexA3 mutation, but the inhibition was not reversed by the recAo254 mutation, indicating a requirement for some other lexA+-regulated gene product in the recA+-independent SDR process. A model is presented for the regulation of the expression of these three types of SDR by the products of the lexA+, rin+ and recF+ genes.
Mol Gen Genet 1987 Jul
PMID:Genetic analysis of constitutive stable DNA replication in rnh mutants of Escherichia coli K12. 282 60

The requirements for the recovery of DNA synthesis in UV-irradiated Escherichia coli were analysed in strains having varied levels of RNase H and RecA protein. We have previously shown (Khidhir et al. 1985) that the recovery of DNA synthesis in E. coli following UV treatment is an inducible SOS function requiring protein synthesis. We proposed that this reflected the need for the synthesis of specific induced replisome reactivation factor(s) for recovery. In this study we now show that recovery of DNA synthesis can in fact take place in the absence of protein synthesis in a mutant lacking RNase H and having high (constitutive) levels of RecA protein. We also show that expression of rnh is inhibited during the SOS response in recA+ but not in a recA- strain. The results are discussed in relation to the mechanism of recovery of DNA synthesis following UV irradiation in E. coli.
Mol Gen Genet 1987 Oct
PMID:Effects of modulation of RNase H production on the recovery of DNA synthesis following UV-irradiation in Escherichia coli. 282 81

Escherichia coli rnh mutants lacking ribonuclease H (RNase H) activity can tolerate deletion of the origin of DNA Replication (delta oriC) and transposon-insertional inactivation of an initiator gene (dnaA::Tn10). Introduction of the recA200 allele encoding a thermolabile RecA protein into rnh- dnaA::Tn10 and rnh- delta oriC mutants strains rendered DNA synthesis and colony formation of these mutants temperature sensitive. The temperature sensitivity and the broth sensitivity (Srm-) of the rnh- dnaA::Tn10 recA200 strain was suppressed by the presence of plasmids (pBR322 derivatives) carrying dnaA+ only when the intact oriC site was present on the chromosome. Lack of RNase H activity neither promoted replication of minichromosomes (pOC24 and p lambda asn20) in the absence of required DnaA+ protein nor inhibited dnaA+-dependent minichromosome replication. These results led to the conclusion that RNase H is not directly involved in the events leading to initiation of DNA replication at oriC. Rather, it functions as a specificity factor by eliminating certain forms of RNA-DNA hybrids which could otherwise be used to prime DNA replication at sites other than oriC.
Mol Gen Genet 1985
PMID:Function of ribonuclease H in initiation of DNA replication in Escherichia coli K-12. 299 5

We have analysed the transcription levels for the convergently overlapping Escherichia coli genes for the DNA polymerase III proofreading function (dnaQ) and ribonuclease H (rnh). The two tandem dnaQ promoters are about three times more active than the single rnh promoter as shown by analysing the level of in vivo transcription using dnaQ-galK and rnh-galK fusions. In E. coli mutants constitutively expressing the pleiotropic SOS response, which includes activities that enhance DNA repair, recombination and mutagenesis, a strong reduction in rnh transcription was observed. The lexA51 recA441 double mutant which fully expresses the SOS response shows the strongest reduction in rnh transcription and the highest increase in dnaQ transcription. Nuclease S1 mapping supported the finding that a constitutive expression of SOS function leads to a strong reduction in rnh transcription.
Mol Gen Genet 1987 Jan
PMID:Reduced transcription of the rnh gene in Escherichia coli mutants expressing the SOS regulon constitutively. 303 43

Mutants of Escherichia coli completely deficient in RNase H activity were isolated by inserting transposon Tn3 into the structural gene for RNase H, rnh, and its promoter. These rnh- mutants exhibited the following phenotypes; (1) the mutants grew fairly normally, (2) rnh- cells could be transformed with ColE1 derivative plasmids, pBR322 and pML21, though the plasmids were relatively unstable, under non selective conditions, (3) rnh- mutations partially suppressed the temperature-sensitive phenotype of plasmid pSC301, a DNA replication initiation mutant derived from pSC101, (4) rnh- mutations suppressed the temperature-sensitive growth character of dnaAts mutant, (5) rnh- cells showed continued DNA synthesis in the presence of chloramphenicol (stable DNA replication). Based on these findings we propose a model for a role of RNase H in the initiation of chromosomal DNA replication. We suggest that two types of RNA primers for initiation of DNA replication are synthesized in a dnaA/oriC-dependent and -independent manner and that only the dnaA/oriC-dependent primer is involved in the normal DNA replication since the dnaA/oriC independent primer is selectively degraded by RNase H.
Mol Gen Genet 1984
PMID:RNase H-defective mutants of Escherichia coli: a possible discriminatory role of RNase H in initiation of DNA replication. 609 45


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