Replacement of the lysine residue in the consensus ATP-binding sequence of the AddA subunit of AddAB drastically affects chromosomal recombination in transformation and transduction of Bacillus subtilis

The ATP-dependent deoxyribonuclease enzyme complex (AddAB) of Bacillus subtilis possesses two consensus ATP-binding sequences, located in the N-terminal region of both subunits. The highly conserved lysine residues in both consensus ATP-binding sequences were replaced by glycine, resulting in the mutant enzyme complexes AddAB-A-K36G (AddA*B) and AddAB-B-K14G (AddAB*). The mutation in subunit AddA reduced DNA repair and chromosomal transformation, and abolished bacteriophage PBS1-mediated transduction. This mutation also resulted in a complete loss of the ATP-dependent exonuclease and helicase activity. In contrast, the mutation in subunit AddB had only marginal effects. The recF and addAB genes are not required for transformation with plasmid DNA, but have overlapping activities in transformation with chromosomal DNA. By contrast to RecF, the AddAB enzyme is essential for PBS1-mediated transduction. However, recF has a more important function with respect to DNA repair than addAB .     

The C terminus of the AddA subunit of the Bacillus subtilis ATP-dependent DNase is required for the ATP-dependent exonuclease activity but not for the helicase activity.

Comparison of subunit AddA of the Bacillus subtilis AddAB enzyme, subunit RecB of the Escherichia coli RecBCD enzyme, and subunit RecB of the Haemophilus influenzae RecBCD enzyme revealed several regions of homology. Whereas the first seven regions are common among helicases, the two C-terminally located regions are unique for RecB of E. coli and H. influenzae and AddA. Deletion of the C-terminal region resulted in the production of an enzyme which showed moderately impaired levels of ATP-dependent helicase activity, whereas the ATP-dependent exonuclease activity was completely destroyed. The mutant enzyme was almost completely capable of complementing E. coli recBCD and B. subtilis addAB strains with respect to DNA repair and homologous recombination. These results strongly suggest that at least part of the C-terminal region of the AddA protein is indispensable for exonuclease activity and that, in contrast to the exonuclease activity, the helicase activity of the addAB gene product is important for DNA repair and homologous recombination.     

A conserved helicase motif of the AddA subunit of the Bacillus subtilis ATP-dependent nuclease (AddAB) is essential for DNA repair and recombination

Various mutations were introduced in a conserved helicase domain (motif VI) of the AddA subunit of the Bacillus subtilis ATP-dependent nuclease (AddAB) by site-directed mutagenesis. These mutations affected the helicase activity and the ATP-dependent exonuclease activity on double-stranded DNA (dsDNA) as the substrate to various degrees, but had hardly any effect on the exonuclease activity on single-stranded DNA (ssDNA), suggesting that exonuclease activity on dsDNA of the enzyme requires unwinding of the DNA. This idea was supported by the finding that, initially, the rate and extent of unwinding of the DNA were higher than those of its degradation to acid-soluble products by the exonucleolytic activity. The effects of the mutations on DNA repair and recombination correlated strongly with their effects on helicase activity. Taken together, these results suggest that motif VI is essential for the helicase activity, and that this activity is required for DNA repair and recombination.     

The AddAB helicase-nuclease catalyses rapid and processive DNA unwinding using a single Superfamily 1A motor domain

The oligomeric state of Superfamily I DNA helicases is the subject of considerable and ongoing debate. While models based on crystal structures imply that a single helicase core domain is sufficient for DNA unwinding activity, biochemical data from several related enzymes suggest that a higher order oligomeric species is required. In this work we characterize the helicase activity of the AddAB helicase-nuclease, which is involved in the repair of double-stranded DNA breaks in Bacillus subtilis. We show that the enzyme is functional as a heterodimer of the AddA and AddB subunits, that it is a rapid and processive DNA helicase, and that it catalyses DNA unwinding using one single-stranded DNA motor of 3' → 5' polarity located in the AddA subunit. The AddB subunit contains a second putative ATP-binding pocket, but this does not contribute to the observed helicase activity and may instead be involved in the recognition of recombination hotspot sequences.     

Helicase-defective RuvB(D113E) promotes RuvAB-mediated branch migration in vitro.

In Escherichia coli, the RuvA and RuvB proteins interact at Holliday junctions to promote branch migration leading to the formation of heteroduplex DNA. RuvA provides junction-binding specificity and RuvB drives ATP-dependent branch migration. Since RuvB contains sequence motifs characteristic of a DNA helicase and RuvAB exhibit helicase activity in vitro, we have analysed the role of DNA unwinding in relation to branch migration. A mutant RuvB protein, RuvB(D113E), mutated in helicase motif II (the DExx box), has been purified to homogeneity. The mutant protein forms hexameric rings on DNA similar to those formed by wild-type protein and promotes branch migration in the presence of RuvA. However, RuvB(D113E) exhibits reduced ATPase activity and is severely compromised in its DNA helicase activity. Models for RuvAB-mediated branch migration that invoke only limited DNA unwinding activity are proposed.     

Mutational analysis of the functional motifs of RuvB, an AAA+ class helicase and motor protein for holliday junction branch migration

Escherichia coli RuvB protein, together with RuvA, promotes branch migration of Holliday junctions during homologous recombination and recombination repair. The RuvB molecular motor is an intrinsic ATP-dependent DNA helicase with a hexameric ring structure and its architecture has been suggested to be related to those of the members of the AAA+ protein class. In this study, we isolated a large number of plasmids carrying ruvB mutant genes and identified amino acid residues important for the RuvB functions by examining the in vivo DNA repair activities of the mutant proteins. Based on these mutational studies and amino acid conservation among various RuvBs, we identified 10 RuvB motifs that agreed well with the features of the AAA+ protein class and that distinguished the primary structure of RuvB from that of typical DNA/RNA helicases with seven conserved helicase motifs.     

Nucleotide sequences and expression in Escherichia coli of the in-phase overlapping Pseudomonas aeruginosa plcR genes.

The translation products of chromosomal DNAs of Pseudomonas aeruginosa encoding phospholipase C (heat-labile hemolysin) have been examined in T7 promoter plasmid vectors and expressed in Escherichia coli cells. A plasmid carrying a 4.7-kilobase (kb) DNA fragment was found to encode the 80-kilodalton (kDa) phospholipase C as well as two more proteins with an apparent molecular mass of 26 and 19 kDa. Expression directed by this DNA fragment with various deletions suggested that the coding region for the two smaller proteins was contained in a 1-kb DNA region. Moreover, the size of both proteins was reduced by the same amount by an internal BglII-BglII DNA deletion, suggesting that they were translated from overlapping genes. Similar results were obtained with another independently cloned 6.1-kb Pseudomonas DNA, which in addition coded for a 31-kDa protein of opposite orientation. The nucleotide sequence of the 1-kb region above revealed an open reading frame with a signal sequence typical of secretory proteins and a potential in-phase internal translation initiation site. Pulse-chase and localization studies in E. coli showed that the 26-kDa protein was a precursor of a secreted periplasmic 23-kDa protein (PlcR1) while the 19-kDa protein (PlcR2) was mostly cytoplasmic. These results indicate the expression of Pseudomonas in-phase overlapping genes in E. coli.     

Effects of lysine-to-glycine mutations in the ATP-binding consensus sequences in the AddA and AddB subunits on the Bacillus subtilis AddAB enzyme activities.

The N-terminal regions of both subunits AddA and AddB of the Bacillus subtilis AddAB enzyme contain amino acid sequences, designated motif I, which are commonly found in ATP-binding enzymes. The functional significance of the motif I regions was studied by replacing the highly conserved lysine residues of the regions in both subunits by glycines and by examination of the resulting mutant enzymes with respect to their enzymatic properties. This study shows that the mutation in subunit AddB hardly affected the ATPase, helicase, and exonuclease activities of the AddAB enzyme. However, the mutation in subunit AddA drastically reduced these activities, as well as the kcat for ATP hydrolysis. The apparent Km for ATP in ATP hydrolysis did not significantly deviate from that of the wild-type enzyme. These results suggest that the lysine residue in motif I of subunit AddA of the AddAB enzyme is not essential for the binding of the nucleotide but has a role in ATP hydrolysis, which is required for the exonuclease and helicase activities of the enzyme.     

rhlB, a new Escherichia coli K-12 gene with an RNA helicase-like protein sequence motif, one of at least five such possible genes in a prokaryote

A newly recognized gene we name rhlB, after RNA helicase-like genes, has been found in the 85-minute region of the Escherichia coli chromosome. This gene encodes protein sequence motifs similar to those known for "D-E-A-D box" gene products. Proteins in this gene family occur in eukaryotes as well as prokaryotes, and, as far as tested, have been found to participate in ATP-dependent RNA helicase or RNA-dependent ATPase activities. The functions of these enzymes are poorly understood. In yeast, mutant phenotypes of various D-E-A-D genes suggest that they function in RNA splicing, processing, or translation. We find that rhlB is necessary for viability only in some genetic backgrounds. Conditional rhlB lethality is not complemented by another E. coli RNA helicase-like gene (srmB). Using primers with homology to consensus sequences in D-E-A-D box proteins, we have recovered DNA fragments amplified from E. coli genomic DNA by polymerase chain reactions. Sequence analysis of these fragments suggests that E. coli possesses at least five RNA helicase-like (rhl) D-E-A-D box genes at widely separated chromosomal locations. The multiplicity of such genes in a prokaryote raises the possibility of important roles for the corresponding class of biologically widespread proteins.     

Structure and function of the Escherichia coli RecE protein, a member of the RecB nuclease domain family

The RecB subunit of the Escherichia coli RecBCD enzyme has both helicase and nuclease activities. The helicase function was localized to an N-terminal domain, whereas the nuclease activity was found in a C-terminal domain. Recent analysis has uncovered a group of proteins that have weak amino acid sequence similarity to the RecB nuclease domain and that are proposed to constitute a family of related proteins (Aravind, L., Walker, D. R., and Koonin, E. V. (1999) Nucleic Acids Res. 27, 1223-1242). One is the E. coli RecE protein (exonuclease VIII), an ATP-independent exonuclease that degrades the 5'-terminated strand of double-stranded DNA. We have made mutations in several residues of RecE that align with the critical residues of RecB, and we find that the mutations reduce or abolish the nuclease activity of RecE but do not affect the enzyme binding to linear double-stranded DNA. Proteolysis experiments with subtilisin show that a stable 34-kilodalton C-terminal domain that contains these critical residues has nuclease activity, whereas no stable proteolytic fragments accumulate from the N-terminal portion of RecE. These results show that RecE has a nuclease domain and active site that are similar to RecB, despite the very weak sequence similarity between the two proteins. These similarities support the hypothesis that the nuclease domains of the two proteins are evolutionarily related.     

Two domains of superfamily I helicases may exist as separate proteins.

DNA and RNA helicases of superfamily I are characterized by seven conserved motifs. The five N-terminal motifs are separated from the two C-terminal ones by a spacer that is highly variable in both sequence and length, suggesting the existence of two distinct domains. Using computer methods for protein sequence analysis, we show that PhoH, an ATP-binding protein that is conserved in Escherichia coli and Mycobacterium leprae, is homologous to the putative N-terminal domain of the helicases, whereas the putative E. coli protein YjhR is homologous to the C-terminal domain. These findings suggest that the N-and C-terminal domains of superfamily I helicases have distinct activities, with only the N-terminal domain having the ATPase activity. It is speculated that PhoH and YjhR have evolved from helicases through deletion of the portions of the helicase genes coding for the C- and N-terminal domain, respectively.     

Escherichia coli SecA helicase activity is not required in vivo for efficient protein translocation or autogenous regulation

SecA is an essential ATP-driven motor protein that binds to preproteins and the translocon to promote protein translocation across the eubacterial plasma membrane. Escherichia coli SecA contains seven conserved motifs characteristic of superfamily II of DNA and RNA helicases, and it has been shown previously to possess RNA helicase activity. SecA has also been shown to be an autogenous repressor that binds to its translation initiation region on secM-secA mRNA, thereby blocking and dissociating 30 S ribosomal subunits. Here we show that SecA is an ATP-dependent helicase that unwinds a mimic of the repressor helix of secM-secA mRNA. Mutational analysis of the seven conserved helicase motifs in SecA allowed us to identify mutants that uncouple SecA-dependent protein translocation activity from its helicase activity. Helicase-defective secA mutants displayed normal protein translocation activity and autogenous repression of secA in vivo. Our studies indicate that SecA helicase activity is nonessential and does not appear to be necessary for efficient protein secretion and secA autoregulation.     

deaD, a new Escherichia coli gene encoding a presumed ATP-dependent RNA helicase, can suppress a mutation in rpsB, the gene encoding ribosomal protein S2.

We have cloned and sequenced a new gene from Escherichia coli which encodes a 64-kDa protein. The inferred amino acid sequence of the protein shows remarkable similarity to eIF4A, a murine translation initiation factor that has an ATP-dependent RNA helicase activity and is a founding member of the D-E-A-D family of proteins (characterized by a conserved Asp-Glu-Ala-Asp motif). Our new gene, called deaD, was cloned as a gene dosage-dependent suppressor of temperature-sensitive mutations in rpsB, the gene encoding ribosomal protein S2. We suggest that the DeaD protein plays a hitherto unknown role in translation in E. coli.     

Cloning and biochemical characterization of Bacillus subtilis YxiN, a DEAD protein specifically activated by 23S rRNA: delineation of a novel sub-family of bacterial DEAD proteins.

DEAD, DEAH and DExH proteins are involved in almost every facet of RNA biochemistry. Members of these protein families exhibit an RNA-dependent ATPase activity and some possess an ATP-dependent RNA helicase activity. Although genetic studies have identified specific functions for certain DEx(D)/(H)proteins from which an RNA substrate can be reasonably inferred, only DbpA from Escherichia coli has been shown to exhibit significant RNA specificity in vitro. Here we describe the characterization of YxiN from Bacillus subtilis, the second DEx(D)/(H)protein to show significant RNA specificity as an isolated, homogenous protein. The ATPase activity of YxiN, like that of DbpA, is stimulated by a 154 nt fragment of 23S rRNA. YxiN has a 2 nM apparent binding constant for this fragment, yet its ATPase activity shows 1800-fold RNA specificity. Along with the conserved motifs shared among all DEAD proteins, YxiN and DbpA have a conserved C-terminal extension. This extension is highly conserved in several additional DEAD proteins. We propose that the C-terminus identifies a protein sub-family whose members bind 23S rRNA and that proteins of this family are likely to function in rRNA maturation/ribosome biogenesis or an unappreciated aspect of translation.     

Purification of RIP60 and RIP100, mammalian proteins with origin-specific DNA-binding and ATP-dependent DNA helicase activities.

Replication of the Chinese hamster dihydrofolate reductase gene (dhfr) initiates near a fragment of stably bent DNA that binds multiple cellular factors. Investigation of protein interactions with the dhfr bent DNA sequences revealed a novel nuclear protein that also binds to domain B of the yeast origin of replication, the autonomously replicating sequence ARS1. The origin-specific DNA-binding activity was purified 9,000-fold from HeLa cell nuclear extract in five chromatographic steps. Protein-DNA cross-linking experiments showed that a 60-kDa polypeptide, which we call RIP60, contained the origin-specific DNA-binding activity. Oligonucleotide displacement assays showed that highly purified fractions of RIP60 also contained an ATP-dependent DNA helicase activity. Covalent radiolabeling with ATP indicated that the DNA helicase activity resided in a 100-kDa polypeptide, RIP100. The cofractionation of an ATP-dependent DNA helicase with an origin-specific DNA-binding activity suggests that RIP60 and RIP100 may be involved in initiation of chromosomal DNA synthesis in mammalian cells.     

Helicobacter pylori AddAB helicase-nuclease and RecA promote recombination-related DNA repair and survival during stomach colonization

Helicobacter pylori colonization of the human stomach is characterized by profound disease-causing inflammation. Bacterial proteins that detoxify reactive oxygen species or recognize damaged DNA adducts promote infection, suggesting that H. pylori requires DNA damage repair for successful in vivo colonization. The molecular mechanisms of repair remain unknown. We identified homologues of the AddAB class of helicase-nuclease enzymes, related to the Escherichia coli RecBCD enzyme, which, with RecA, is required for repair of DNA breaks and homologous recombination. H. pylori mutants lacking addA or addB genes lack detectable ATP-dependent nuclease activity, and the cloned H. pylori addAB genes restore both nuclease and helicase activities to an E. coli recBCD deletion mutant. H. pylori addAB and recA mutants have a reduced capacity for stomach colonization. These mutants are sensitive to DNA damaging agents and have reduced frequencies of apparent gene conversion between homologous genes encoding outer membrane proteins. Our results reveal requirements for double-strand break repair and recombination during both acute and chronic phases of H. pylori stomach infection.     

Leuconostoc lactis beta-galactosidase is encoded by two overlapping genes.

A 16-kb BamHI fragment of the lactose plasmid pNZ63 from Leuconostoc lactis NZ6009 was cloned in Escherichia coli MC1061 by using pACYC184 and was found to express a functional beta-galactosidase. Deletion and complementation analysis showed that the coding region for beta-galactosidase was located on a 5.8-kb SalI-BamHI fragment. Nucleotide sequence analysis demonstrated that this fragment contained two partially overlapping genes, lacL (1,878 bp) and lacM (963 bp), that could encode proteins with calculated sizes of 72,113 and 35,389 Da, respectively. The L. lactis beta-galactosidase was overproduced in E. coli by using a lambda pL expression system. Two new proteins with M(r)s of 75,000 and 36,000 appeared upon induction of PL. The N-terminal sequences of these proteins corresponded to those deduced from the lacL and lacM gene sequences. Mutation and deletion analysis showed that lacL expression is essential for LacM production and that both the lacL and lacM genes are required for the production of a functional beta-galactosidase in E. coli. The deduced amino acid sequences of the LacL and LacM proteins showed considerable identity with the sequences of the N- and C-terminal parts, respectively, of beta-galactosidases from other lactic acid bacteria or E. coli. DNA and protein sequence alignments suggest that the L. lactis lacL and lacM genes have been generated by an internal deletion in an ancestral beta-galactosidase gene.     

Molecular cloning of cDNA encoding human DNA helicase Q1 which has homology to Escherichia coli Rec Q helicase and localization of the gene at chromosome 12p12.

A complementary DNA encoding DNA-dependent ATPase Q1 possessing DNA helicase activity, which is the major DNA-dependent ATPase in human cell extracts, was cloned from a cDNA library of human KB cells. The predicted amino acid sequence has seven consecutive motifs conserved in the RNA and DNA helicase super family and DNA helicase Q1 belongs to DEXH helicase family. A homology search indicated that helicase Q1 had 47% homology in the seven conserved regions with Escherichia coli RecQ protein. Three RNA bands of 4.0, 3.3, and 2.2 kilobases were detected in HeLa cells by Northern blotting. Analysis of the genomic DNA indicated the presence of a homologous gene in mouse cells. The DNA helicase Q1 gene was localized on the short arm of human chromosome 12 at 12p12.     

Molecular cloning of structural and regulatory hydrogenase (hox) genes of Alcaligenes eutrophus H16

A gene bank of the 450-kilobase (kb) megaplasmid pHG1 from the hydrogen-oxidizing bacterium Alcaligenes eutrophus H16 was constructed in the broad-host-range mobilizable vector pSUP202 and maintained in Escherichia coli. hox DNA was identified by screening the E. coli gene bank for restoration of hydrogenase activity in A. eutrophus Hox mutants. Hybrid plasmids that contained an 11.6-kb EcoRI fragment restored soluble NAD-dependent hydrogenase activity when transferred by conjugation into one class of Hos- mutants. An insertion mutant impaired in particulate hydrogenase was partially restored in Hop activity by an 11-kb EcoRI fragment. A contiguous sequence of two EcoRI fragments of 8.6 and 2.0 kb generated Hox+ recombinants from mutants that were devoid of both hydrogenase proteins. hox DNA was subcloned into the vector pVK101. The resulting recombinant plasmids were used in complementation studies. The results indicate that we have cloned parts of the structural genes coding for Hos and Hop activity and a complete regulatory hox DNA sequence which encodes the thermosensitive, energy-dependent derepression signal of hydrogenase synthesis in A. eutrophus H16.