Exonuclease VII was first identified in 1974 as a DNA exonuclease that did not require any divalent cations for activity. Indeed, Escherichia coli ExoVII was identified in partially purified extracts in the presence of EDTA. ExoVII is comprised of two subunits (XseA and XseB) that are highly conserved and present in most sequenced prokaryotic genomes, but are not seen in eukaryotes. To better understand this exonuclease family, we have characterized an ExoVII homolog from Thermotoga maritima. Thermotoga maritima XseA/B homologs TM1768 and TM1769 were co-expressed and purified, and show robust nuclease activity at 80°C. This activity is magnesium dependent and is inhibited by phosphate ions, which distinguish it from E. coli ExoVII. Nevertheless, both E. coli and T. maritima ExoVII share a similar putative active site motif with two conserved aspartate residues in the large (XseA/TM1768) subunit. We show that these residues, Asp235 and Asp240, are essential for the nuclease activity of T. maritima ExoVII. We hypothesize that the ExoVII family of nucleases can be sub-divided into two sub-families based on EDTA resistance and that T. maritima ExoVII is the first member of the branch that is characterized by EDTA sensitivity and inhibition by phosphate.
Strains of Escherichia coli containing reduced levels of exonuclease VII activity due to mutations in the xseB gene have been isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Seven mutants of independent origin deficient in exonuclease VII activity were obtained. Four of these contained defects in xseA, a locus which has been previously identified, and three others contained mutations in a gene distinct from xseA, which we have designated xseB. Genetic mapping studies place the xseB locus between proC and dnaZ. Exonuclease VII purified from KLC835 (xseA+ xseB3) is more heat labile than enzyme purified from the parent strain PA610 (xse+), showing that xseB is a structural gene for exonuclease VII. The isolation of lambda transducing phage carrying xseA is also described.
Mutants of Escherichia coli having reduced levels of exonuclease VII activity have been isolated by a mass screening procedure. Nine mutants, five of which are known to be of independent origin, were obtained and designated xse. The defects in these strains lie at two or more loci. One of these loci, xseA, lies in the interval between purG and purC; it is 93 to 97% co-transducible with guaA. The order of the genes in this region is purG-xseA guaA,B-purC. The available data do not allow xseA to be ordered with respect to guaA,B. Exonuclease VII purified from E. coli KLC3 xseA3 is more heat labile than exonuclease VII purified from the parent, E. coli PA610 xse+. Therefore, xseA is the structural gene for exonuclease VII. Mutants with defects in the xseA gene show increased sensitivity to nalidixic acid and have an abnormally high frequency of recombination (hyper-Rec phenotype) as measured by the procedure of Konrad and Lehlman (1974). The hyper-Rec character of xseA strains is approximately one-half that of the polAex1 mutant defective in the 5' leads to 3' hydrolytic activity of deoxyribonucleic acid polymerase I. The double mutant, polAex1 xseA7, is twice as hyper-Rec as the polAex1 mutant alone. The xseA- strains are slightly more sensitive to ultraviolet irradiation than the parent strain. Bacteriophages T7, fd, and lambdared grow normally in xseA- strains.
A series of Escherichia coli strains deficient in the 5'----3' exonuclease activity associated with deoxyribonucleic acid (DNA) polymerase I (exonuclease VI) and exonuclease VII has been constructed. Both of these enzymes are capable of pyrimidine dimer excision in vitro. These strains were examined for conditional lethality, sensitivity to ultraviolet (UV) and X-irradiation, postirradiation DNA degradation, and ability to excise pyrimidine dimers. It was found that strains deficient in both exonuclease VI (polAex-) and exonuclease VII (xseA-) are significantly reduced in their ability to survive incubation at elevated temperature (43 degrees C) beyond the reduction previously observed for the polAex single mutants. The UV and X-ray sensitivity of the exonuclease VI-deficient strains was not increased by the addition of the xseA7 mutation. Mutants deficient in both enzymes are about as efficient as wild-type strains at excising dimers produced by up to 40 J/m2 UV. At higher doses strains containing only polAex- mutations show reduced ability to excise dimers; however, the interpretation of dimer excision data at these doses is complicated by extreme postirradiation DNA degradation in these strains. The additional deficiency in the polAex xseA7 double-mutant strains has no significant effect on either postirradiation DNA degradation or the apparent deficiency in dimer excision at high UV doses observed in polAex single mutants.
In vitro, the methyl-directed mismatch repair system of Escherichia coli requires the single-strand exonuclease activity of either ExoI, ExoVII, or RecJ and possibly a fourth, unknown single-strand exonuclease. We have created the first precise null mutations in genes encoding ExoI and ExoVII and find that cells lacking these nucleases and RecJ perform mismatch repair in vivo normally such that triple-null mutants display normal mutation rates. ExoI, ExoVII, and RecJ are either redundant with another function(s) or are unnecessary for mismatch repair in vivo.
Escherichia coli strains carrying null alleles of genes encoding single-strand-specific exonucleases ExoI and ExoVII display elevated frameshift mutation rates but not base substitution mutation rates. We characterized increased spontaneous frameshift mutation in ExoI− ExoVII− cells and report that some of this effect requires RecA, an inducible SOS DNA damage response, and the low-fidelity, SOS-induced DNA polymerase DinB/PolIV, which makes frameshift mutations preferentially. We also find that SOS is induced in ExoI− ExoVII− cells. The data imply a role for the single-stranded exonucleases in guarding the genome against mutagenesis by removing excess single-stranded DNA that, if left, leads to SOS induction and PolIV-dependent mutagenesis. Previous results implicated PolIV in E. coli mutagenesis specifically during starvation or antibiotic stresses. Our data imply that PolIV can also promote mutation in growing cells under genome stress due to excess single-stranded DNA.
To assess the contributions of single-strand DNases (ssDNases) to recombination in a recBCD+ background, we studied 31 strains with all combinations of null alleles of exonuclease I (Δxon), exonuclease VII (xseA), RecJ DNase (recJ), and SbcCD DNase (sbcCD) and exonuclease I mutant alleles xonA2 and sbcB15. The xse recJ sbcCD Δxon and xse recJ sbcCD sbcB15 quadruple mutants were cold sensitive, while the quadruple mutant with xonA2 was not. UV sensitivity increased with ssDNase deficiencies. Most triple and quadruple mutants were highly sensitive. The absence of ssDNases hardly affected P1 transductional recombinant formation, and conjugational recombinant production was decreased (as much as 94%) in several cases. Strains with sbcB15 were generally like the wild type. We determined that the sbcB15 mutation caused an A183V exchange in exonuclease motif III and identified xonA2 as a stop codon eliminating the terminal 8 amino acids. Purified enzymes had 1.6% (SbcB15) and 0.9% (XonA2) of the specific activity of wild-type Xon (Xon+), respectively, with altered activity profiles. In gel shift assays, SbcB15 associated relatively stably with 3′ DNA overhangs, giving protection against Xon+. In addition to their postsynaptic roles in the RecBCD pathway, exonuclease I and RecJ are proposed to have presynaptic roles of DNA end blunting. Blunting may be specifically required during conjugation to make DNAs with overhangs RecBCD targets for initiation of recombination. Evidence is provided that SbcB15 protein, known to activate the RecF pathway in recBC strains, contributes independently of RecF to recombination in recBCD+ cells. DNA end binding by SbcB15 can also explain other specific phenotypes of strains with sbcB15.
Lambda Red recombineering is a powerful technique for making targeted genetic changes in bacteria. However, many applications are limited by the frequency of recombination. Previous studies have suggested that endogenous nucleases may hinder recombination by degrading the exogenous DNA used for recombineering. In this work, we identify ExoVII as a nuclease which degrades the ends of single-stranded DNA (ssDNA) oligonucleotides and double-stranded DNA (dsDNA) cassettes. Removing this nuclease improves both recombination frequency and the inheritance of mutations at the 3′ ends of ssDNA and dsDNA. Extending this approach, we show that removing a set of five exonucleases (RecJ, ExoI, ExoVII, ExoX, and Lambda Exo) substantially improves the performance of co-selection multiplex automatable genome engineering (CoS-MAGE). In a given round of CoS-MAGE with ten ssDNA oligonucleotides, the five nuclease knockout strain has on average 46% more alleles converted per clone, 200% more clones with five or more allele conversions, and 35% fewer clones without any allele conversions. Finally, we use these nuclease knockout strains to investigate and clarify the effects of oligonucleotide phosphorothioation on recombination frequency. The results described in this work provide further mechanistic insight into recombineering, and substantially improve recombineering performance.
We have investigated in vivo the role of the carboxy-terminal domain of the Bacillus subtilis Single-Stranded DNA Binding protein (SSBCter) as a recruitment platform at active chromosomal forks for many proteins of the genome maintenance machineries. We probed this SSBCter interactome using GFP fusions and by Tap-tag and biochemical analysis. It includes at least 12 proteins. The interactome was previously shown to include PriA, RecG, and RecQ and extended in this study by addition of DnaE, SbcC, RarA, RecJ, RecO, XseA, Ung, YpbB, and YrrC. Targeting of YpbB to active forks appears to depend on RecS, a RecQ paralogue, with which it forms a stable complex. Most of these SSB partners are conserved in bacteria, while others, such as the essential DNA polymerase DnaE, YrrC, and the YpbB/RecS complex, appear to be specific to B. subtilis. SSBCter deletion has a moderate impact on B. subtilis cell growth. However, it markedly affects the efficiency of repair of damaged genomic DNA and arrested replication forks. ssbΔCter mutant cells appear deficient in RecA loading on ssDNA, explaining their inefficiency in triggering the SOS response upon exposure to genotoxic agents. Together, our findings show that the bacterial SSBCter acts as a DNA maintenance hub at active chromosomal forks that secures their propagation along the genome.
Cell multiplication relies primarily on the complete and accurate duplication of the genome. Thus, all organisms have evolved multiple mechanisms to protect, repair, and re-activate the DNA replication forks. A large body of research is currently aimed at deciphering the mechanisms that precisely direct the proteins involved in these rescue pathways towards the chromosome replication forks. Here, we have used the model bacterium Bacillus subtilis to demonstrate that the active chromosomal DNA replication forks are pre-equipped with many such rescue effectors via their direct physical interaction with the carboxy-terminal end (Cter) of the Single-Stranded DNA Binding protein (SSB). A detailed analysis of the multiple defects of viable B. subtilis mutants deleted for the Cter of SSB (SSBCter) revealed the vital role of this domain for the maintenance of genome integrity and fork propagation. The inability to grow at high temperature is a major defect of the ssbΔCter mutant. We show that this lethality can be specifically suppressed by overexpression of RecO, one of the numerous partners of SSB, apparently by mediating the loading of the RecA recombinase on ssDNA.
The transcription initiation site for rat 45S precursor ribosomal RNA synthesis was determined by nuclease protection mapping with two single-strand endonucleases. S1 and mung bean, and one single-strand exonuclease, ExoVII. These experiments were performed with end-labeled ribosomal DNA from double-stranded pBR322 recombinants and from single-stranded M13 recombinants. Results from experiments using both kinds of DNA and all three enzymes showed that the 5' end of 45S RNA mapped to a unique site 125 bases upstream from the Hind III site in the ribosomal DNA gene. The DNA surrounding this site (designated +1) was sequenced from -281 to +641. The entire sequence of this region shows extensive homology to the comparable region of mouse. This includes three stretches of T residues in the non-coding strand between +300 and +630. Two sets of direct repeats adjacent to these T-rich regions are observed. Comparison of the mouse and human ribosomal DNA transcription initiation sites with the rat sequence reported in this paper demonstrates a conserved sequence at +2 to +16, CTGACACGCTGTCCT. This suggests that this region may be important for the initiation of transcription on mammalian ribosomal DNAs.
Little is known about factors which enable Salmonella serotypes to circulate within populations of livestock and domestic fowl. We have identified a DNA region which is present in Salmonella serotypes commonly isolated from livestock and domestic fowl (S. enterica subspecies I) but absent from reptile-associated Salmonella serotypes (S. bongori and S. enterica subspecies II to VII). This DNA region was cloned from Salmonella serotype Typhimurium and sequence analysis revealed the presence of a 6,105-bp open reading frame, designated shdA, whose product's deduced amino acid sequence displayed homology to that of AIDA-I from diarrheagenic Escherichia coli, MisL of serotype Typhimurium, and IcsA of Shigella flexneri. The shdA gene was located adjacent to xseA at 52 min, in a 30-kb DNA region which is not present in Escherichia coli K-12. A serotype Typhimurium shdA mutant was shed with the feces in reduced numbers and for a shorter period of time compared to its isogenic parent. A possible role for the shdA gene during the expansion in host range of S. enterica subspecies I to include warm-blooded vertebrates is discussed.
Nutritional competence is the ability of bacterial cells to utilize exogenous double-stranded DNA molecules as a nutrient source. We previously identified several genes in Escherichia coli that are important for this process and proposed a model, based on models of natural competence and transformation in bacteria, where it is assumed that single-stranded DNA (ssDNA) is degraded following entry into the cytoplasm. Since E. coli has several exonucleases, we determined whether they play a role in the long-term survival and the catabolism of DNA as a nutrient. We show here that mutants lacking either ExoI, ExoVII, ExoX, or RecJ are viable during all phases of the bacterial life cycle yet cannot compete with wild-type cells during long-term stationary-phase incubation. We also show that nuclease mutants, alone or in combination, are defective in DNA catabolism, with the exception of the ExoX− single mutant. The ExoX− mutant consumes double-stranded DNA better than wild-type cells, possibly implying the presence of two pathways in E. coli for the processing of ssDNA as it enters the cytoplasm.
Human exonuclease 1 (hExo1) plays important roles in DNA repair and recombination processes that maintain genomic integrity. It is a member of the 5′ structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. We present structures of hExo1 in complex with a DNA substrate, followed by mutagenesis studies, and propose a common mechanism by which this nuclease family recognizes and processes diverse DNA structures. hExo1 induces a sharp bend in the DNA at nicks or gaps. Frayed 5′ ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exo-nucleolytic processing. Conformational control of a mobile region in the catalytic site suggests a mechanism for allosteric regulation by binding to protein partners. The relative arrangement of substrate binding sites in these enzymes provides an elegant solution to a complex geometrical puzzle of substrate recognition and processing.
Temperature induction of an Escherichia coli strains with lambda cI1857 integrated in the guaB gene has been used to produce strains containing chromosomal deletions extending into the xse and upp genes. By utilizing strains containing these deletions, it has been possible to order the genes in the guanine operon with respect to the xseA and upp genes. The order of the genes in this region is glyA-hisS-xseA-guaO-guaB-guaA-purG-upp-purC.
We have constructed a strain of Escherichia coli that is defective in exonuclease VII and uracil-DNA glycosylase activities. This strain (xse ung) facilitates the quantitation of single-stranded apurinic-apyrimidinic endonuclease activity in crude extracts. Quantitative comparisons of single-stranded apurinic-apyrimidinic endonuclease activity under conditions in which uvrC protein is overexpressed showed no differences, suggesting that single-stranded apurinic-apyrimidinic endonuclease and uvrC protein are probably distinct.
In Drosophila, XX embryos are fated to develop as females, and XY embryos as males, because the diplo-X dose of four X-linked signal element genes, XSEs, activates the Sex-lethal establishment promoter, SxlPe, whereas the haplo-X XSE dose leaves SxlPe off. The threshold response of SxlPe to XSE concentrations depends in part on the bHLH repressor, Deadpan, present in equal amounts in XX and XY embryos. We identified canonical and non-canonical DNA-binding sites for Dpn at SxlPe and found that cis-acting mutations in the Dpn-binding sites caused stronger and earlier Sxl expression than did deletion of dpn implicating other bHLH repressors in Sxl regulation. Maternal Hey encodes one such bHLH regulator but the E(spl) locus does not. Elimination of the maternal corepressor Groucho also caused strong ectopic Sxl expression in XY, and premature Sxl activation in XX embryos, but Sxl was still expressed differently in the sexes. Our findings suggest that Groucho and associated maternal and zygotic bHLH repressors define the threshold XSE concentrations needed to activate SxlPe and that they participate directly in sex signal amplification. We present a model in which the XSE signal is amplified by a feedback mechanism that interferes with Gro-mediated repression in XX, but not XY embryos.
Hes; X:A ratio; genetic switch; helix-loop-helix; scute; repression; WRPW; X chromosome-counting
In bacteria, double-strand DNA break (DSB) repair involves an exonuclease/helicase (exo/hel) and a short regulatory DNA sequence (Chi) that attenuates exonuclease activity and stimulates DNA repair. Despite their key role in cell survival, these DSB repair components show surprisingly little conservation. The best-studied exo/hel, RecBCD of Escherichia coli, is composed of three subunits. In contrast, RexAB of Lactococcus lactis and exo/hel enzymes of other low-guanine-plus-cytosine branch gram-positive bacteria contain two subunits. We report that RexAB functions via a novel mechanism compared to that of the RecBCD model. Two potential nuclease motifs are present in RexAB compared with a single nuclease in RecBCD. Site-specific mutagenesis of the RexA nuclease motif abolished all nuclease activity. In contrast, the RexB nuclease motif mutants displayed strongly reduced nuclease activity but maintained Chi recognition and had a Chi-stimulated hyperrecombination phenotype. The distinct phenotypes resulting from RexA or RexB nuclease inactivation lead us to suggest that each of the identified active nuclease sites in RexAB is involved in the degradation of one DNA strand. In RecBCD, the single RecB nuclease degrades both DNA strands and is presumably positioned by RecD. The presence of two nucleases would suggest that this RecD function is dispensable in RexAB.
Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII–colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage λ exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and Aquifex. Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including McrA and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the λ exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.
The PD-(D/E)xK superfamily, containing a wide variety of other exo- and endonucleases, is a notable example of general function conservation in the face of extreme sequence and structural variation. Almost all members employ a small number of shared conserved residues to bind catalytically essential metal ions and thereby effect DNA cleavage. The crystal structure of the RecBCD prokaryotic DNA repair machinery shows that RecB contains such a nuclease domain at its C-terminus. The RecC C-terminal region was reported as having a novel fold.
The RecC C-terminal region can be divided into an alpha/beta domain and a smaller alpha-helical bundle domain. Here we show that the alpha/beta domain is homologous to the RecB nuclease domain but lacks the features necessary for catalysis. Instead, the domain has a novel function within the nuclease superfamily – providing a hoop through which single-stranded DNA passes. Comparison with other structures of nuclease domains bound to DNA reveals strikingly different modes of ligand binding. The alpha-helical bundle domain contributes the pin which splits the DNA duplex.
The demonstrated homology of RecB and RecC shows how evolution acted to produce the present RecBCD complex through aggregation of new domains as well as functional divergence and structural redeployment of existing domains. Distantly homologous nuclease(-like) domains bind DNA in highly diverse manners.
Adenoviruses are nonenveloped viruses with an ∼36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin α, importin β, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.
Escherichia coli Exonuclease IX (ExoIX), encoded by the xni gene, was the first identified member of a novel subfamily of ubiquitous flap endonucleases (FENs), which possess only one of the two catalytic metal-binding sites characteristic of other FENs. We have solved the first structure of one of these enzymes, that of ExoIX itself, at high resolution in DNA-bound and DNA-free forms. In the enzyme–DNA cocrystal, the single catalytic site binds two magnesium ions. The structures also reveal a binding site in the C-terminal domain where a potassium ion is directly coordinated by five main chain carbonyl groups, and we show this site is essential for DNA binding. This site resembles structurally and functionally the potassium sites in the human FEN1 and exonuclease 1 enzymes. Fluorescence anisotropy measurements and the crystal structures of the ExoIX:DNA complexes show that this potassium ion interacts directly with a phosphate diester in the substrate DNA.
Human Exo1 is a member of the RAD2 nuclease family with roles in replication, repair and recombination. Despite sharing significant amino acid sequence homology, the RAD2 proteins exhibit disparate nuclease properties and biological functions. In order to identify elements that dictate substrate selectivity within the RAD2 family, we sought to identify residues key to Exo1 nuclease activity and to characterize the molecular details of the human Exo1–DNA interaction. Site-specific mutagenesis studies demonstrate that amino acids D78, D173 and D225 are critical for Exo1 nuclease function. In addition, we show that the chemical nature of the 5′-terminus has a major impact on Exo1 nuclease efficiency, with a 5′-phosphate group stimulating degradation 10-fold and a 5′-biotin inhibiting degradation 10-fold (relative to a 5′-hydroxyl moiety). An abasic lesion located within a substrate DNA strand impedes Exo1 nucleolytic degradation, and a 5′-terminal abasic residue reduces nuclease efficiency 2-fold. Hydroxyl radical footprinting indicates that Exo1 binds predominantly along the minor groove of flap DNA, downstream of the junction. As will be discussed, our results favor the notion that the single-stranded DNA structure is pinched by the helical arch of the protein and not threaded through this key recognition loop. Furthermore, our studies indicate that significant, presumably biologically relevant, differences exist between the active site dynamics of Exo1 and Fen1.
Exonuclease 1 (EXO1) and Flap endonuclease 1 (FEN1) are members of the RAD2 family of structure-specific nucleases. Genetic analysis has identified roles for EXO1 and FEN1 in replication, recombination, DNA repair and maintenance of telomeres. Telomeres are composed of G-rich repeats that readily form G4 DNA. We recently showed that human EXO1 and FEN1 exhibit distinct activities on G4 DNA substrates representative of intermediates in immunoglobulin class switch recombination.
We have now compared activities of these enzymes on telomeric substrates bearing G4 DNA, identifying non-overlapping functions that provide mechanistic insight into the distinct telomeric phenotypes caused by their deficiencies. We show that hFEN1 but not hEXO1 cleaves substrates bearing telomeric G4 DNA 5′-flaps, consistent with the requirement for FEN1 in telomeric lagging strand replication. Both hEXO1 and hFEN1 are active on substrates bearing telomeric G4 DNA tails, resembling uncapped telomeres. Notably, hEXO1 but not hFEN1 is active on transcribed telomeric G-loops.
Our results suggest that EXO1 may act at transcription-induced telomeric structures to promote telomere recombination while FEN1 has a dominant role in lagging strand replication at telomeres. Both enzymes can create ssDNA at uncapped telomere ends thereby contributing to recombination.
Type IIS restriction endonuclease Eco31I is a ‘short-distance cutter’, which cleaves DNA strands close to its recognition sequence, 5′-GGTCTC(1/5). Previously, it has been proposed that related endonucleases recognizing a common sequence core GTCTC possess two active sites for cleavage of both strands in the DNA substrate. Here, we present bioinformatic identification and experimental evidence for a single nuclease active site. We identified a short region of homology between Eco31I and HNH nucleases, constructed a three-dimensional model of the putative catalytic domain and validated our predictions by random and site-specific mutagenesis. The restriction mechanism of Eco31I is suggested by analogy to the mechanisms of phage T4 endonuclease VII and homing endonuclease I-PpoI. We propose that residues D311 and N334 coordinate the cofactor. H312 acts as a general base activating water molecule for the nucleophilic attack. K337 together with R340 and D345 are located in close proximity to the active center and are essential for correct folding of catalytic motif, while D345 together with R264 and D273 could be directly involved in DNA binding. We also predict that the Eco31I catalytic domain contains a putative Zn-binding site, which is essential for its structural integrity. Our results suggest that the HNH-like active site is involved in the cleavage of both strands in the DNA substrate. On the other hand, analysis of site-specific mutants in the region, previously suggested to harbor the second active site, revealed its irrelevance to the nuclease activity. Thus, our data argue against the earlier prediction and indicate the presence of a single conserved active site in Type IIS restriction endonucleases that recognize common sequence core GTCTC.
restriction endonuclease; Type IIS; HNH; endonuclease VII; active site
The WRN gene defective in the premature aging disorder Werner syndrome encodes a helicase/exonuclease. We examined the ability of WRN to rescue DNA damage sensitivity of a yeast mutant defective in the Rad50 subunit of Mre11-Rad50- Xrs2 nuclease complex implicated in homologous recombination repair. Genetic studies revealed WRN operates in a yEXO1-dependent pathway to rescue rad50 sensitivity to methylmethane sulfonate (MMS) and prevent mitotic catastrophe. WRN helicase, but not exonuclease, is required for MMS resistance. WRN missense mutations in helicase or RecQ C-terminal domains interfered with the ability of WRN to rescue rad50 MMS sensitivity. WRN does not rescue rad50 ionizing radiation (IR) sensitivity, suggesting that WRN, in collaboration with yEXO1, is tailored to relieve replicational stress imposed by alkylated base damage. WRN and yEXO1 are associated with each other in vivo. Purified WRN stimulates hEXO1 nuclease activity on DNA substrates associated with a stalled or regressed replication fork. We propose WRN helicase operates in an EXO1-dependent pathway to help cells survive replicational stress. In contrast to WRN, BLM helicase defective in Bloom’s syndrome failed to rescue rad50 MMS sensitivity, but partially restored IR resistance, suggesting a delineation of function by the human RecQ helicases.
Werner syndrome; helicase; RecQ; rad50; replicational stress; genomic instability