Bloom syndrome protein forms an oligomeric ring structure and belongs to a group of DNA helicases showing extensive homology to the
Escherichia coli DNA helicase RecQ, a suppressor of illegitimate ...recombination. After over-production in
E.
coli
, we have purified the RecQ core of BLM consisting of the DEAH, RecQ-Ct and HRDC domains (amino acid residues 642–1290). The BLM
642–1290 fragment could function as a DNA-stimulated ATPase and as a DNA helicase, displaying the same substrate specificity as the full-size protein. Gel-filtration experiments revealed that BLM
642–1290 exists as a monomer both in solution and in its single-stranded DNA-bound form, even in the presence of Mg
2+ and ATPγS. Rates of ATP hydrolysis and DNA unwinding by BLM
642–1290 showed a hyperbolic dependence on ATP concentration, excluding a co-operative interaction between ATP-binding sites. Using a λ Spi
− assay, we have found that the BLM
642–1290 fragment is able to partially substitute for the RecQ helicase in suppressing illegitimate recombination in
E.
coli
. A deletion of 182 C-terminal amino acid residues of BLM
642–1290, including the HRDC domain, resulted in helicase and single-stranded DNA-binding defects, whereas kinetic parameters for ATP hydrolysis of this mutant were close to the BLM
642–1290 values. This confirms the prediction that the HRDC domain serves as an auxiliary DNA-binding domain. Mutations at several conserved residues within the RecQ-Ct domain of BLM reduced ATPase and helicase activities severely as well as single-stranded DNA-binding of the enzyme. Together, these data define a minimal helicase domain of BLM and demonstrate its ability to act as a suppressor of illegitimate recombination.
ObjectivesTo investigate if centre-specific levels of perinatal interventional activity were associated with neonatal and neurodevelopmental outcome at 2 years of age in two separately analysed ...cohorts of infants: cohort A born at 22–25 and cohort B born at 26–27 gestational weeks, respectively.DesignGeographically defined, retrospective cohort study.SettingAll nine level III perinatal centres (neonatal intensive care units and affiliated obstetrical services) in Switzerland.PatientsAll live-born infants in Switzerland in 2006–2013 below 28 gestational weeks, excluding infants with major congenital malformation.Outcome measuresOutcomes at 2 years corrected for prematurity were mortality, survival with any major neonatal morbidity and with severe-to-moderate neurodevelopmental impairment (NDI).ResultsCohort A associated birth in a centre with high perinatal activity with low mortality adjusted OR (aOR 0.22; 95% CI 0.16 to 0.32), while no association was observed with survival with major morbidity (aOR 0.74; 95% CI 0.46 to 1.19) and with NDI (aOR 0.97; 95% CI 0.46 to 2.02). Median age at death (8 vs 4 days) and length of stay (100 vs 73 days) were higher in high than in low activity centres. The results for cohort B mirrored those for cohort A.ConclusionsCentres with high perinatal activity in Switzerland have a significantly lower risk for mortality while having comparable outcomes among survivors. This confirms the results of other studies but in a geographically defined area applying a more restrictive approach to initiation of perinatal intensive care than previous studies. The study adds that infants up to 28 weeks benefited from a higher perinatal activity and why further research is required to better estimate the added burden on children who ultimately do not survive.
DNA cleavage by type III restriction endonucleases requires two inversely oriented asymmetric recognition sequences and results from ATP-dependent DNA translocation and collision of two enzyme ...molecules. Here, we characterized the structure and mode of action of the related EcoP1I and EcoP15I enzymes. Analytical ultracentrifugation and gel quantification revealed a common Res2Mod2 subunit stoichiometry. Single alanine substitutions in the putative nuclease active site of ResP1 and ResP15 abolished DNA but not ATP hydrolysis, whilst a substitution in helicase motif VI abolished both activities. Positively supercoiled DNA substrates containing a pair of inversely oriented recognition sites were cleaved inefficiently, whereas the corresponding relaxed and negatively supercoiled substrates were cleaved efficiently, suggesting that DNA overtwisting impedes the convergence of the translocating enzymes. EcoP1I and EcoP15I could co-operate in DNA cleavage on circular substrate containing several EcoP1I sites inversely oriented to a single EcoP15I site; cleavage occurred predominantly at the EcoP15I site. EcoP15I alone showed nicking activity on these molecules, cutting exclusively the top DNA strand at its recognition site. This activity was dependent on enzyme concentration and local DNA sequence. The EcoP1I nuclease mutant greatly stimulated the EcoP15I nicking activity, while the EcoP1I motif VI mutant did not. Moreover, combining an EcoP15I nuclease mutant with wild-type EcoP1I resulted in cutting the bottom DNA strand at the EcoP15I site. These data suggest that double-strand breaks result from top strand cleavage by a Res subunit proximal to the site of cleavage, whilst bottom strand cleavage is catalysed by a Res subunit supplied in trans by the distal endonuclease in the collision complex.
Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non‐specific cleavage site. We have examined several potential blocks to DNA ...translocation, such as positive supercoiling or a Holliday junction, for their ability to trigger DNA cleavage by type I restriction enzymes. Introduction of positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzyme's ability to select cleavage sites randomly throughout the DNA molecule. Thus, positive supercoiling does not prevent DNA translocation. EcoR124II endonuclease cleaved DNA at Holliday junctions present on both linear and negatively supercoiled substrates. The latter substrate was cleaved by a single enzyme molecule at two sites, one on either side of the junction, consistent with a bi‐directional translocation model. Linear DNA molecules with two recognition sites for endonucleases from different type I families were cut between the sites when both enzymes were added simultaneously but not when a single enzyme was added. We propose that type I restriction enzymes can track along a DNA substrate irrespective of its topology and cleave DNA at any barrier that is able to halt the translocation process.
DNA cleavage by the type III restriction endonuclease
EcoP1I was analysed on circular and catenane DNA in a variety of buffers with different salts. In the presence of the cofactor
S-adenosyl ...methionine (AdoMet), and irrespective of buffer, only substrates with two
EcoP1I sites in inverted repeat were susceptible to cleavage. Maximal activity was achieved at a Res
2Mod
2 to site ratio of ∼1:1 yet resulted in cleavage at only one of the two sites. In contrast, the outcome of reactions in the absence of AdoMet was dependent upon the identity of the monovalent buffer components, in particular the identity of the cation. With Na
+, cleavage was observed only on substrates with two sites in inverted repeat at elevated enzyme to site ratios (>15:1). However, with K
+ every substrate tested was susceptible to cleavage above an enzyme to site ratio of ∼3:1, including a DNA molecule with two directly repeated sites and even a DNA molecule with a single site. Above an enzyme to site ratio of 2:1, substrates with two sites in inverted repeat were cleaved at both cognate sites. The rates of cleavage suggested two separate events: a fast primary reaction for the first cleavage of a pair of inverted sites; and an order-of-magnitude slower secondary reaction for the second cleavage of the pair or for the first cleavage of all other site combinations.
EcoP1I enzymes mutated in either the ATPase or nuclease motifs did not produce the secondary cleavage reactions. Thus, AdoMet appears to play a dual role in type III endonuclease reactions: Firstly, as an allosteric activator, promoting DNA association; and secondly, as a “specificity factor”, ensuring that cleavage occurs only when two endonucleases bind two recognition sites in a designated orientation. However, given the right conditions, AdoMet is not strictly required for DNA cleavage by a type III enzyme.
Although the DNA cleavage mechanism of Type I restriction–modification enzymes has been extensively studied, the mode of cleavage remains elusive. In this work, DNA ends produced by EcoKI, EcoAI and ...EcoR124I, members of the Type IA, IB and IC families, respectively, have been characterized by cloning and sequencing restriction products from the reactions with a plasmid DNA substrate containing a single recognition site for each enzyme. Here, we show that all three enzymes cut this substrate randomly with no preference for a particular base composition surrounding the cleavage site, producing both 5′- and 3′-overhangs of varying lengths. EcoAI preferentially generated 3′-overhangs of 2–3 nt, whereas EcoKI and EcoR124I displayed some preference for the formation of 5′-overhangs of a length of ∼6–7 and 3–5 nt, respectively. A mutant EcoAI endonuclease assembled from wild-type and nuclease-deficient restriction subunits generated a high proportion of nicked circular DNA, whereas the wild-type enzyme catalyzed efficient cleavage of both DNA strands. We conclude that Type I restriction enzymes require two restriction subunits to introduce DNA double-strand breaks, each providing one catalytic center for phosphodiester bond hydrolysis. Possible models for DNA cleavage are discussed.
Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation past the enzyme. During this reaction, the enzyme ...remains bound to the recognition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visualised by electron microscopy. To further investigate the mechanism of DNA translocation by type I restriction enzymes, we have probed the reaction intermediates with DNA topoisomerases. A DNA cleavage-deficient mutant of
EcoAI, which has normal DNA translocation and ATPase activities, was used in these DNA supercoiling assays. In the presence of eubacterial DNA topoisomerase I, which specifically removes negative supercoils, the
EcoAI mutant introduced positive supercoils into relaxed plasmid DNA substrate in a reaction dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type
EcoAI endonuclease. Positive supercoils were not seen when eubacterial DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I, which removes both positive and negative supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to the product of the supercoiling reaction resulted in its rapid relaxation. These results are consistent with a model in which
EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates positive supercoils ahead and negative supercoils behind the moving complex in the contracting and expanding DNA loops, respectively. In addition, we show that the highly positively supercoiled DNA generated by the
EcoAI mutant is cleaved by
EcoAI wild-type endonuclease much more slowly than relaxed DNA. This suggests that the topological changes in the DNA substrate associated with DNA translocation by type I restriction enzymes do not appear to be the trigger for DNA cleavage.
The DNA specificity subunit (HsdS) of type I restriction-modification enzymes is composed of two independent target recognition domains and several regions whose amino acid sequence is conserved ...within an enzyme family. The conserved regions participate in intersubunit interactions with two modification subunits (HsdM) and two restriction subunits (HsdR) to form the complete endonuclease. It has been proposed that the domains of the HsdS subunit have a circular organisation providing the required symmetry for their interaction with the other subunits and with the bipartite DNA target. To test this model, we circularly permuted the HsdS subunit of the type IB R-M enzyme
EcoAI at the DNA level by direct linkage of codons for original termini and introduction of new termini elsewhere along the N-terminal and central conserved regions. By analysing the activity of mutant enzymes, two circularly permuted variants of HsdS that had termini located at equivalent positions in the N-terminal and central repeats, respectively, were found to fold into a functional DNA recognition subunit with wild-type specificity, suggesting a close proximity of the N and C termini in the native protein. The wild-type HsdS subunit was purified to homogeneity and shown to form a stable trimeric complex with HsdM, M
2S
1, which was fully active as a DNA methyltransferase. Gel electrophoretic mobility shift assays revealed that the HsdS protein alone was not able to form a specific complex with a 30-mer oligoduplex containing a single
EcoAI recognition site. However, addition of stoichiometric amounts of HsdM to HsdS led to efficient specific DNA binding. Our data provide evidence for the circular organisation of domains of the HsdS subunit. In addition, they suggest a possible role of HsdM subunits in the formation of this structure.
Type I restriction enzymes bind to specific DNA sequences but subsequently translocate nonspecific DNA past the complex in a reaction coupled to ATP hydrolysis and cleave DNA at any barrier that can ...halt the translocation process. The restriction subunit of these enzymes, HsdR, contains a cluster of seven amino acid sequence motifs typical of heli-case superfamily II, that are believed to be relevant to the ATP-dependent DNA translocation. Alignment of all available HsdR sequences reveals an additional conserved region at the protein N-terminus with a consensus sequence reminiscent of the P-D…(D/E)-X-K catalytic motif of many type II restriction enzymes. To investigate the role of these conserved residues, we have produced mutants of the type IB restriction enzyme EcoAI. We have found that single alanine substitutions at Asp-61, Glu-76 and Lys-78 residues of the HsdR subunit abolished the enzyme's restriction activity but had no effect on its ATPase and DNA translocation activities, suggesting that these residues are part of the active site for DNA cleavage.
Type III restriction/modification systems recognize short non‐palindromic sequences, only one strand of which can be methylated. Replication of type III‐modified DNA produces completely unmethylated ...recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation‐specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site‐specific, but EcoP15I‐modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme‐to‐site ratio, in that site‐saturating enzyme levels elicit cleavage exclusively between the closest pair of head‐to‐head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I‐DNA complexes.
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