Search tips
Search criteria

Results 1-25 (798808)

Clipboard (0)

Related Articles

1.  Identification of two conserved aspartic acid residues required for DNA digestion by a novel thermophilic Exonuclease VII in Thermotoga maritima 
Nucleic Acids Research  2008;36(18):5992-6003.
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.
PMCID: PMC2566859  PMID: 18812402
2.  Isolation and preliminary characterization of Escherichia coli mutants deficient in exonuclease VII. 
Journal of Bacteriology  1983;155(3):1116-1122.
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.
PMCID: PMC217806  PMID: 6350262
3.  Escherichia coli mutants deficient in exonuclease VII. 
Journal of Bacteriology  1977;129(2):934-947.
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.
PMCID: PMC235032  PMID: 320198
4.  Effects of Single-Strand DNases ExoI, RecJ, ExoVII, and SbcCD on Homologous Recombination of recBCD+ Strains of Escherichia coli and Roles of SbcB15 and XonA2 ExoI Mutant Enzymes▿  
Journal of Bacteriology  2007;190(1):179-192.
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.
PMCID: PMC2223745  PMID: 17965170
5.  Deoxyribonucleic acid repair in Escherichia coli mutants deficient in the 5'----3' exonuclease activity of deoxyribonucleic acid polymerase I and exonuclease VII. 
Journal of Bacteriology  1977;130(2):667-675.
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.
PMCID: PMC235266  PMID: 400786
6.  Mismatch Repair in Escherichia coli Cells Lacking Single-Strand Exonucleases ExoI, ExoVII, and RecJ 
Journal of Bacteriology  1998;180(4):989-993.
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.
PMCID: PMC106982  PMID: 9473057
7.  The DNA exonucleases of Escherichia coli 
EcoSal Plus  2011;4(2):10.1128/ecosalplus.4.4.7.
DNA exonucleases, enzymes that hydrolyze phosphodiester bonds in DNA from a free end, play important cellular roles in DNA repair, genetic recombination and mutation avoidance in all organisms. This article reviews the structure, biochemistry and biological functions of the 17 exonucleases currently identified in the bacterium Escherichia coli. These include the exonucleases associated with DNA polymerases I (polA), II (polB) and III (dnaQ/mutD), Exonucleases I (xonA/sbcB), III (xthA), IV, VII (xseAB), IX (xni/xgdG) and X (exoX), the RecBCD, RecJ, and RecE exonucleases, SbcCD endo/exonuclease, the DNA exonuclease activities of RNase T (rnt) and Endonuclease IV (nfo) and TatD. These enzymes are diverse in terms of substrate specificity and biochemical properties and have specialized biological roles. Most of these enzymes fall into structural families with characteristic sequence motifs, and members of many of these families can be found in all domains of life.
PMCID: PMC4238392  PMID: 26442508
DNase; DNA repair; mutation; homologous recombination; genome rearrangements; DNA polymerase; abasic sites; mismatch repair; proofreading; double-strand break repair; dsDNA; ssDNA; metal binding; mutator; lagging strand; flap endonuclease; base excision repair; replication gap repair; tandem repeats
8.  The C-Terminal Domain of the Bacterial SSB Protein Acts as a DNA Maintenance Hub at Active Chromosome Replication Forks 
PLoS Genetics  2010;6(12):e1001238.
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.
Author Summary
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.
PMCID: PMC3000357  PMID: 21170359
9.  Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases 
PLoS ONE  2012;7(9):e44638.
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.
PMCID: PMC3434165  PMID: 22957093
10.  Single-Strand-Specific Exonucleases Prevent Frameshift Mutagenesis by Suppressing SOS Induction and the Action of DinB/DNA Polymerase IV in Growing Cells 
Journal of Bacteriology  2006;188(7):2336-2342.
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.
PMCID: PMC1428391  PMID: 16547019
11.  Identification and sequence of the initiation site for rat 45S ribosomal RNA synthesis. 
Nucleic Acids Research  1983;11(10):3317-3332.
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.
PMCID: PMC325965  PMID: 6304628
12.  Induction of Lung GSH and Glutamate Cysteine Ligase by 1,4-phenylenebis(methylene)selenocyanate and its Glutathione Conjugate: Role of Nuclear factor-erythroid 2-Related Factor 2 
Free radical biology & medicine  2012;52(10):2064-2071.
The synthetic organoselenium agent 1,4- phenylenebis(methylene)selenocyanate (p-XSC) and its glutathione (GSH) conjugate (p-XSeSG), are potent chemopreventive agents in several preclinical models. p-XSC is also an effective inducer of GSH in mouse lung. Our objectives were to test the hypothesis that GSH induction by p-XSC occurs through upregulation of the rate-limiting GSH biosynthetic enzyme glutamate cysteine ligase (GCL), through activation of antioxidant response elements (ARE) in GCL genes via activation of nuclear factor-erythroid 2-related factor 2 (Nrf2). p-XSC feeding (10 ppm Se) increased GSH (230%) and upregulated the catalytic subunit of GCL (GCLc) (55%), extracellular related kinase (ERK) (220%) and nuclear Nrf2 (610%) in lung but not liver after 14 days in the rat (P<0.05). Similarly, p-XSeSG feeding (10 ppm) induced lung GCLc (88%) and GSH (200%) (P<0.05), while the naturally-occurring selenomethionine had no effect. Both p-XSC and p-XSeSG activated a luciferase reporter in HepG2 ARE reporter cells up to 3-fold for p-XSC and ≥5-fold for p-XSeSG. Luciferase activation by p-XSeSG was associated with enhanced levels of GSH, GCLc and nuclear Nrf2, which were significantly reduced by co-incubation with short interfering RNA targeting Nrf2 (siNrf2). The dependence of GCL induction on Nrf2 was confirmed in Nrf2 deficient mouse embryonic fibroblasts (MEF) where p-XSeSG induced GCL subunits in wildtype, but not Nrf2 deficient cells (p<0.05). These results indicate that p-XSC may act through the Nrf2 pathway in vivo, and that p-XSeSG is the putative metabolite responsible for such activation, thus offering p-XSeSG as a less toxic, yet highly efficacious inducer of GSH.
PMCID: PMC3475320  PMID: 22542796
Organoselenium; p-XSC; p-XSeSG; glutathione; γ-GCL; Nrf2; MEF; antioxidant response element (ARE); HepG2-ARE; Lung cancer; Fisher 344 rat
13.  A Role for Single-Stranded Exonucleases in the Use of DNA as a Nutrient▿  
Journal of Bacteriology  2009;191(11):3712-3716.
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.
PMCID: PMC2681894  PMID: 19329645
14.  Maternal Groucho and bHLH repressors amplify the dose-sensitive X chromosome signal in Drosophila sex determination 
Developmental biology  2008;323(2):248-260.
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.
PMCID: PMC2653429  PMID: 18773886
Hes; X:A ratio; genetic switch; helix-loop-helix; scute; repression; WRPW; X chromosome-counting
15.  Peptide inhibitors identify roles for SSB C-terminal residues in SSB/Exonuclease I complex formation† 
Biochemistry  2009;48(29):6764-6771.
Bacterial single-stranded (ss) DNA-binding proteins (SSBs) facilitate DNA replication, recombination, and repair processes in part by recruiting diverse genome maintenance enzymes to ssDNA. This function utilizes the C-terminus of SSB (SSB-Ct) as a common binding site for SSB’s protein partners. The SSB-Ct is a highly conserved, amphipathic sequence, comprising acidic and hydrophobic elements. A crystal structure of E. coli Exonuclease I (ExoI) bound to a peptide comprising the E. coli SSB-Ct sequence shows that the C-terminal-most SSB-Ct Phe anchors the peptide to a binding pocket on ExoI and implicates electrostatic binding roles for the acidic SSB-Ct residues. Here, we use SSB-Ct peptide variants in competition experiments to examine the roles of individual SSB-Ct residues in binding ExoI in solution. Altering the C-terminal-most Pro or Phe residues in the SSB-Ct strongly impairs SSB-Ct binding to ExoI, confirming a major role for the hydrophobic SSB-Ct residues in binding ExoI. Alteration of N-terminal SSB-Ct residues leads to changes that reflect cumulative electrostatic binding roles for the Asp residues in SSB-Ct. The SSB-Ct peptides also abrogate SSB stimulation of ExoI activity through a competitive inhibition mechanism, indicating that the peptides can disrupt ExoI/SSB/ssDNA ternary complexes. Differences in the potency of the SSB-Ct peptide variants in the binding and nuclease inhibition studies indicate that the acidic SSB-Ct residues play a more prominent role in the context of the ternary complex than in the minimal ExoI/SSB-Ct interaction. Together, these data identify roles for residues in the SSB-Ct that are important for SSB complex formation with its protein partners.
PMCID: PMC2746433  PMID: 19527069
16.  Molecular interactions of human Exo1 with DNA 
Nucleic Acids Research  2002;30(4):942-949.
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.
PMCID: PMC100345  PMID: 11842105
17.  The shdA Gene Is Restricted to Serotypes of Salmonella enterica Subspecies I and Contributes to Efficient and Prolonged Fecal Shedding 
Infection and Immunity  2000;68(5):2720-2727.
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.
PMCID: PMC97480  PMID: 10768965
18.  Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks 
DNA repair  2010;10(1):73-86.
Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the subnuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal (788QIKLNELW795). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390–490 and 787–846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.
PMCID: PMC4586255  PMID: 20970388
hEXO1; PCNA; Replication foci; Double strand break; DNA mismatch repair
19.  An inactivated nuclease-like domain in RecC with novel function: implications for evolution 
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.
PMCID: PMC1185551  PMID: 15985153
20.  Hydrolytic function of Exo1 in mammalian mismatch repair 
Nucleic Acids Research  2014;42(11):7104-7112.
Genetic and biochemical studies have previously implicated exonuclease 1 (Exo1) in yeast and mammalian mismatch repair, with results suggesting that function of the protein in the reaction depends on both its hydrolytic activity and its ability to interact with other components of the repair system. However, recent analysis of an Exo1-E109K knockin mouse has concluded that Exo1 function in mammalian mismatch repair is restricted to a structural role, a conclusion based on a prior report that N-terminal His-tagged Exo1-E109K is hydrolytically defective. Because Glu-109 is distant from the nuclease hydrolytic center, we have compared the activity of untagged full-length Exo1-E109K with that of wild type Exo1 and the hydrolytically defective active site mutant Exo1-D173A. We show that the activity of Exo1-E109K is comparable to that of wild type enzyme in a conventional exonuclease assay and that in contrast to a D173A active site mutant, Exo1-E109K is fully functional in mismatch-provoked excision and repair. We conclude that the catalytic function of Exo1 is required for its participation in mismatch repair. We also consider the other phenotypes of the Exo1-E109K mouse in the context of Exo1 hydrolytic function.
PMCID: PMC4066806  PMID: 24829455
21.  Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis 
Nucleic Acids Research  2011;39(22):9803-9819.
Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5′-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg2+ or Mn2+ ions. 5′-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a ‘doughnut-shaped’ toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg2+ ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.
PMCID: PMC3239189  PMID: 21893587
22.  Biochemical relationships between the 53-kilodalton (Exo53) and 49-kilodalton (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa. 
Journal of Bacteriology  1997;179(5):1609-1613.
Genetic studies have shown that the 53-kDa (Exo53) and 49-kDa (ExoS) forms of exoenzyme S of Pseudomonas aeruginosa are encoded by separate genes, termed exoT and exoS, respectively. Although ExoS and Exo53 possess 76% primary amino acid homology, Exo53 has been shown to express ADP-ribosyltransferase activity at about 0.2% of the specific activity of ExoS. The mechanism for the lower ADP-ribosyltransferase activity of Exo53 relative to ExoS was analyzed by using a recombinant deletion protein which contained the catalytic domain of Exo53, comprising its 223 carboxyl-terminal residues (termed N223-53). N223-53 was expressed in Escherichia coli as a stable, soluble fusion protein which was purified to >80% homogeneity. Under linear velocity conditions, N223-53 catalyzed the FAS (for factor activating exoenzyme S)-dependent ADP-ribosylation of soybean trypsin inhibitor (SBTI) at 0.4% and of the Ras protein at 1.0% of the rates of catalysis by N222-49. N222-49 is a protein comprising the 222 carboxyl-terminal residues of ExoS, which represent its catalytic domain. N223-53 possessed binding affinities for NAD and SBTI similar to those of N222-49 (less than fivefold differences in Kms) but showed a lower velocity rate for the ADP-ribosylation of SBTI. This indicated that the primary defect for ADP-ribosylation by Exo53 resided within its catalytic capacity. Analysis of hybrid proteins, composed of reciprocal halves of N223-53 and N222-49, localized the catalytic defect to residues between positions 235 and 349 of N223-53. E385 was also identified as a potential active site residue of Exo53.
PMCID: PMC178873  PMID: 9045820
23.  Exo1 phosphorylation status controls the hydroxyurea sensitivity of cells lacking the Pol32 subunit of DNA polymerases delta and zeta 
DNA repair  2014;24:26-36.
Exo1 belongs to the Rad2 family of structure-specific nucleases and possesses 5′-3′ exonuclease activity on double-stranded DNA substrates. Exo1 interacts physically with the DNA mismatch repair (MMR) proteins Msh2 and Mlh1 and is involved in the excision of the mispaired nucleotide. Independent of its role in MMR, Exo1 contributes to long-range resection of DNA double-strand break (DSB) ends to facilitate their repair by homologous recombination (HR), and was recently identified as a component of error-free DNA damage tolerance pathways. Here we show that Exo1 activity increases the hydroxyurea sensitivity of cells lacking Pol32, a subunit of DNA polymerases δ and ζ Both, phospho-mimicking and dephospho-mimicking exo1 mutants act as hypermorphs, as evidenced by an increase in HU sensitivity of pol32Δ cells, suggesting that they are trapped in an active form and that phosphorylation of Exo1 at residues S372, S567, S587, S692 is necessary, but insufficient, for the accurate regulation of Exo1 activity at stalled replication forks. In contrast, neither phosphorylation status is important for Exo1's role in MMR or in the suppression of genome instability in cells lacking Sgs1 helicase. This ability of an EXO1 deletion to suppress the HU hypersensitivity of pol32Δ cells is in contrast to the negative genetic interaction between deletions of EXO1 and POL32 in MMS-treated cells as well as the role of EXO1 in DNA-damage treated rad53 and mec1 mutants.
PMCID: PMC4418189  PMID: 25457771
Pol32; Exo1; Exo1 phosphorylation; gross-chromosomal rearrangements; hydroxyurea hypersensitivity
24.  14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif 
DNA repair  2012;11(3):267-277.
Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies.
PMCID: PMC4586177  PMID: 22222486
hEXO1; 14-3-3; DNA repair; Cell cycle control; DNA damage; Protein phosphorylation
25.  Antimutator Alleles of Yeast DNA Polymerase Gamma Modulate the Balance between DNA Synthesis and Excision 
PLoS ONE  2011;6(11):e27847.
Mutations in mitochondrial DNA (mtDNA) are an important cause of disease and perhaps aging in human. DNA polymerase gamma (pol γ ), the unique replicase inside mitochondria, plays a key role in the fidelity of mtDNA replication through selection of the correct nucleotide and 3′-5′ exonuclease proofreading. For the first time, we have isolated and characterized antimutator alleles in the yeast pol γ (Mip1). These mip1 mutations, localised in the 3′-5′ exonuclease and polymerase domains, elicit a 2–15 fold decrease in the frequency of mtDNA point mutations in an msh1-1 strain which is partially deficient in mtDNA mismatch-repair. In vitro experiments show that in all mutants the balance between DNA synthesis and exonucleolysis is shifted towards excision when compared to wild-type, suggesting that in vivo more opportunity is given to the editing function for removing the replicative errors. This results in partial compensation for the mismatch-repair defects and a decrease in mtDNA point mutation rate. However, in all mutants but one the antimutator trait is lost in the wild-type MSH1 background. Accordingly, the polymerases of selected mutants show reduced oligonucleotide primed M13 ssDNA synthesis and to a lesser extent DNA binding affinity, suggesting that in mismatch-repair proficient cells efficient DNA synthesis is required to reach optimal accuracy. In contrast, the Mip1-A256T polymerase, which displays wild-type like DNA synthesis activity, increases mtDNA replication fidelity in both MSH1 and msh1-1 backgrounds. Altogether, our data show that accuracy of wild-type Mip1 is probably not optimal and can be improved by specific (often conservative) amino acid substitutions that define a pol γ area including a loop of the palm subdomain, two residues near the ExoII motif and an exonuclease helix-coil-helix module in close vicinity to the polymerase domain. These elements modulate in a subtle manner the balance between DNA polymerization and excision.
PMCID: PMC3218072  PMID: 22114710

Results 1-25 (798808)