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PubMed1996

Effects of Xenopus laevis mitochondrial single-stranded DNA-binding protein on primer-template binding and 3'-->5' exonuclease activity of DNA polymerase gamma.

Mitochondrial DNA (mtDNA) is replicated by DNA polymerase gamma by a strand displacement mechanism involving mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB stimulates the overall rate of DNA synthesis on singly-primed M13 DNA mainly by stimulating the processivity of DNA synthesis rather than by stimulating primer recognition. We used electrophoretic mobility shift methods to study the effects of mtSSB on primer-template recognition by DNA pol gamma. Preliminary experiments showed that single mtSSB tetramers bind tightly to oligo(dT) single strands containing 32 to 48 residues. An oligonucleotide primer-template was designed with an 18-mer primer annealed to the 3'-portion of a 71-mer template containing 40 dT residues at its 5'-end as a binding site for mtSSB. DNA pol gamma bound to this primer-template either in the absence or presence of mtSSB in complexes that remained intact and enzymatically active following native gel electrophoresis. Association of mtSSB with the 5'-dT40-tail in the 18:71-mer primer-template reduced the binding of DNA polymerase gamma and the efficiency of primer extension. Binding of mtSSB to single-stranded DNA was also observed to block the action of the 3'-->5' exonuclease of DNA polymerase gamma. The size of fragments protected from 3'-->5' exonuclease trimming increases with increasing ionic strength in a manner consistent with the known salt dependence of the binding site size of Escherichia coli SSB.

Animals↗
PubMed1996

Inhibition of the 3' --> 5' exonuclease of human DNA polymerase epsilon by fludarabine-terminated DNA.

Incorporation of the anticancer drug fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate; F-ara-AMP) into the 3'-end of DNA during replication causes termination of DNA strand elongation and is strongly correlated with loss of clonogenicity. Because the proofreading mechanisms that remove 3'-F-ara-AMP from DNA represent a possible means of resistance to the drug, the present study investigated the excision of incorporated F-ara-AMP from DNA by the 3' --> 5'-exonuclease activity of DNA polymerase epsilon from human leukemia CEM cells. Using the drug-containing and normal deoxynucleotide oligomers (21-base) annealed to M13mp18(+) DNA as the excision substrates, we demonstrated that DNA polymerase epsilon was unable to effectively remove F-ara-AMP from the 3'-end of the oligomer. However, 3'-terminal dAMP and subsequently other deoxynucleotides were readily excised from DNA in a distributive fashion. Kinetic evaluation demonstrated that although DNA polymerase epsilon has a higher affinity for F-ara-AMP-terminated DNA (Km = 7.1 pM) than for dAMP-terminated DNA of otherwise identical sequence (Km = 265 pM), excision of F-ara-AMP proceeded at a substantially slower rate (Vmax = 0.053 pmol/min/mg) than for 3'-terminal dAMP (Vmax = 1.96 pmol/min/mg). When the 3'-5' phosphodiester bond between F-ara-AMP at the 3'-terminus and the adjacent normal deoxynucleotide was cleaved by DNA polymerase epsilon, the reaction products appeared to remain associated with the enzyme but without the formation of a covalent bond. No further excision of the remaining oligomers was observed after the addition of fresh DNA polymerase epsilon to the reaction. Furthermore, the addition of DNA polymerase alpha and deoxynucleoside triphosphates to the excision reaction failed to extend the oligomers. After DNA polymerase epsilon had been incubated with 3'-F-ara-AMP-21-mer for 10 min, the enzyme was no longer able to excise 3'-terminal dAMP from a freshly added normal 21-mer annealed to M13mp18(+) template. We conclude that the 3' --> 5' exonuclease of human DNA polymerase epsilon can remove 3'-terminal F-ara-AMP from DNA with difficulty and that this excision results in a mechanism-mediated formation of "dead end complex."

Base Sequence↗
PubMed1999

Variation in the steady state kinetic parameters of wild type and mutant T5 5'-3'-exonuclease with pH. Protonation of Lys-83 is critical for DNA binding.

T5 5'-3'-exonuclease is a member of a family of homologous 5'-nucleases essential for DNA replication and repair. We have measured the variation of the steady state parameters of the enzyme with pH. The log of the association constant of the enzyme and substrate is pH-independent between pH 5 and 7, but at higher pH, it decreases (gradient -0.91 +/- 0.1) with increasing pH. The log of the turnover number increases (gradient 0.9 +/- 0.01) with increasing pH until a pH-independent plateau is reached. The T5 5'-3'-exonuclease-catalyzed reaction requires the protonation of a single residue for substrate binding, whereas kcat depends on a single deprotonation as demonstrated by the bell-shaped dependence of log (kcat/Km) on pH. To investigate the role of a conserved lysine (Lys-83), the pH profile of log (kcat/Km) of a K83A mutant was determined and found to increase with pH (gradient 1.01 +/- 0. 01) until a pH-independent plateau is reached. We therefore conclude that protonation of Lys-83 in the wild type protein facilitates DNA binding. The origin of the pH dependence of the kcat parameter of the wild type enzyme is discussed.

Bacteriophages↗
PubMed2000

A 54-kDa fragment of the Poly(A)-specific ribonuclease is an oligomeric, processive, and cap-interacting Poly(A)-specific 3' exonuclease.

We have previously identified a HeLa cell 3' exonuclease specific for degrading poly(A) tails of mRNAs. Here we report on the purification and identification of a calf thymus 54-kDa polypeptide associated with a similar 3' exonuclease activity. The 54-kDa polypeptide was shown to be a fragment of the poly(A)-specific ribonuclease 74-kDa polypeptide. The native molecular mass of the nuclease activity was estimated to be 180-220 kDa. Protein/protein cross-linking revealed an oligomeric structure, most likely consisting of three subunits. The purified nuclease activity released 5'-AMP as the reaction product and degraded poly(A) in a highly processive fashion. The activity required monovalent cations and was dependent on divalent metal ions. The RNA substrate requirement was investigated, and it was found that the nuclease was highly poly(A)-specific and that only 3' end-located poly(A) was degraded by the activity. RNA substrates capped with m(7)G(5')ppp(5')G were more efficiently degraded than noncapped RNA substrates. Addition of free m(7)G(5')ppp(5')G cap analogue inhibited poly(A) degradation in vitro, suggesting a functional link between the RNA 5' end cap structure and poly(A) degradation at the 3' end of the RNA.

Adenosine Monophosphate↗
PubMed2000

Poly(A) tail-dependent exonuclease AtRrp41p from Arabidopsis thaliana rescues 5.8 S rRNA processing and mRNA decay defects of the yeast ski6 mutant and is found in an exosome-sized complex in plant and yeast cells.

Eukaryotic 3'-->5' exonucleolytic activities are essential for a wide variety of reactions of RNA maturation and metabolism, including processing of rRNA, small nuclear RNA, and small nucleolar RNA, and mRNA decay. Two related but distinct forms of a complex containing 10 3'-->5' exonucleases, the exosome, are found in yeast nucleus and cytoplasm, respectively, and related complexes exist in human cells. Here we report on the characterization of the AtRrp41p, an Arabidopsis thaliana homolog of the Saccharomyces cerevisiae exosome subunit Rrp41p (Ski6p). Purified recombinant AtRrp41p displays a processive phosphorolytic exonuclease activity and requires a single-stranded poly(A) tail on a substrate RNA as a "loading pad." The expression of the Arabidopsis RRP41 cDNA in yeast rescues the 5.8 S rRNA processing and 3'-->5' mRNA degradation defects of the yeast ski6-100 mutant. However, neither of these defects can explain the conditional lethal phenotype of the ski6-100 strain. Importantly, AtRrp41p shares additional function(s) with the yeast Rrp41p which are essential for cell viability because it also rescues the rrp41 (ski6) null mutant. AtRrp41p is found predominantly in a high molecular mass complex in Arabidopsis and in yeast cells, and it interacts in vitro with the yeast Rrp44p and Rrp4p exosome subunits, suggesting that it can participate in evolutionarily conserved interactions that could be essential for the integrity of the exosome complex.

Arabidopsis↗
PubMed2001

The interaction of DNA mismatch repair proteins with human exonuclease I.

Exonucleolytic degradation of DNA is an essential part of many DNA metabolic processes including DNA mismatch repair (MMR) and recombination. Human exonuclease I (hExoI) is a member of a family of conserved 5' --> 3' exonucleases, which are implicated in these processes by genetic studies. Here, we demonstrate that hExoI binds strongly to hMLH1, and we describe interaction regions between hExoI and the MMR proteins hMSH2, hMSH3, and hMLH1. In addition, hExoI forms an immunoprecipitable complex with hMLH1/hPMS2 in vivo. The study of interaction regions suggests a biochemical mechanism of the involvement of hExoI as a downstream effector in MMR and/or DNA recombination.

Adaptor Proteins, Signal Transducing↗
PubMed2003

The exonucleolytic and endonucleolytic cleavage activities of human exonuclease 1 are stimulated by an interaction with the carboxyl-terminal region of the Werner syndrome protein.

Exonuclease 1 (EXO-1), a member of the RAD2 family of nucleases, has recently been proposed to function in the genetic pathways of DNA recombination, repair, and replication which are important for genome integrity. Although the role of EXO-1 is not well understood, its 5' to 3'-exonuclease and flap endonuclease activities may cleave intermediates that arise during DNA metabolism. In this study, we provide evidence that the Werner syndrome protein (WRN) physically interacts with human EXO-1 and dramatically stimulates both the exonucleolytic and endonucleolytic incision functions of EXO-1. The functional interaction between WRN and EXO-1 is mediated by a protein domain of WRN which interacts with flap endonuclease 1 (FEN-1). Thus, the genomic instability observed in WRN-/- cells may be at least partially attributed to the lack of interactions between the WRN protein and human nucleases including EXO-1.

Binding Sites↗
PubMed2006

Concerted action of exonuclease and Gap-dependent endonuclease activities of FEN-1 contributes to the resolution of triplet repeat sequences (CTG)n- and (GAA)n-derived secondary structures formed during maturation of Okazaki fragments.

There is much evidence to indicate that FEN-1 efficiently cleaves single-stranded DNA flaps but is unable to process double-stranded flaps or flaps adopting secondary structures. However, the absence of Fen1 in yeast results in a significant increase in trinucleotide repeat (TNR) expansion. There are then two possibilities. One is that TNRs do not always form stable secondary structures or that FEN-1 has an alternative approach to resolve the secondary structures. In the present study, we test the hypothesis that concerted action of exonuclease and gap-dependent endonuclease activities of FEN-1 play a role in the resolution of secondary structures formed by (CTG)n and (GAA)n repeats. Employing a yeast FEN-1 mutant, E176A, which is deficient in exonuclease (EXO) and gap endonuclease (GEN) activities but retains almost all of its flap endonuclease (FEN) activity, we show severe defects in the cleavage of various TNR intermediate substrates. Precise knock-in of this point mutation causes an increase in both the expansion and fragility of a (CTG)n tract in vivo. Taken together, our biochemical and genetic analyses suggest that although FEN activity is important for single-stranded flap processing, EXO and GEN activities may contribute to the resolution of structured flaps. A model is presented to explain how the concerted action of EXO and GEN activities may contribute to resolving structured flaps, thereby preventing their expansion in the genome.

Alanine↗
PubMed1992

Using lambda exonuclease inhibition assays to map carcinogen binding sites.

Previous restriction mapping studies (M.A. Mallamaci, D.P. Reed and S.A. Winkle, J. Biomolecular Structure and Dynamics, in press (1992)) have indicated that a small number of locations on the plasmid pBR322 may be high affinity binding sites for the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF). PBR322 was reacted with acetoxyAAF to produce DNA with one, three or seven acetoxyAAF moieties per DNA molecule. Thus only the higher affinity binding sites are affected. Subsequent digestion with the restriction enzyme Hinf I produced fragments containing previously indicated locations of potential acetoxyAAF binding sites. Fragments thought not to contain binding sites were also examined as controls. The isolated fragments, singly 32P end-labeled, were digested with lambda exonuclease. The three fragments suspected of containing acetoxyAAF binding sites possess new lambda exonuclease inhibition sites when the fragments are obtained from acetoxyAAF reacted DNA. No such inhibition sites are found with the two fragments suggested previously not to contain acetoxyAAF binding sites. These carcinogen produced inhibition sites occur in sequences which are similar, suggesting that acetoxyAAF preferentially may target a small number of sequences.

Acetoxyacetylaminofluorene↗
PubMed1987

Use of lambda exonuclease for efficient oligonucleotide-mediated site-directed deletion and point mutation of double-stranded DNA.

A novel approach to oligonucleotide-mediated, site-directed in vitro mutagenesis is described that allows for the efficient generation of sequence modifications on double-stranded substrates without the need for subcloning into special vectors. Site-directed deletions as well as point mutations were introduced into the genes encoding human tissue plasminogen activator (tPA) and the Bacillus amyloliquefaciens alpha-amylase gene using lambda exonuclease to enzymatically degrade DNA 5' to 3' in order to generate a single-stranded template in the immediate vicinity of the oligonucleotide annealing site. The mutagenizing oligonucleotide, used both to redefine the 5' end of the molecule and to introduce base changes, was annealed to the single-stranded target sequence producing substrates for both the exonucleolytic and polymerizing activities of DNA polymerase Klenow fragment. Resolution of the resultant heteroduplex by Escherichia coli resulted in the generation of the desired deletion point mutation in the tPA sequence with an efficiency of 38% as determined by differential hybridization and 32% as determined by restriction analysis, with final verification by sequence data. As a further test of the method, two point mutations were introduced simultaneously with the desired sequence deletion into the Bacillus amyloliquefaciens alpha-amylase gene, generating a Pst I restriction site at the junction of the DNA encoding the signal peptide and the mature enzyme with an efficiency of 0.3% as determined by sequence data of hybridization-positive/Pst I-positive clones. The lambda exonuclease procedure is designed for use in situations where site-directed deletions must be introduced efficiently alone or with single or double point mutations.

Bacillus↗
PubMed1996

Antisense 2'-O-methyloligoribonucleotides hybridized to RNA block a nuclear, ATP-dependent 3'-5' exonuclease.

RNA hybridized to 2'-O-methyloligoribonucleotides and incubated in nuclear extracts from HeLa cells is truncated, resulting in a distinct product terminated at the 5' end of the antisense oligonucleotide. The activity responsible for this effect is not RNase H but rather a novel exonuclease degrading RNA in the 3' to 5' direction. The enzymes requires ATP and Mg2+ ions. Except for dATP, no other nucleoside triphosphate or nonhydrolyzable ATP analog supports the exonucleolytic activity. In spite of the nuclear origin and activity requirements similar to those required for pre-mRNA splicing, the exonuclease operates with equal efficiency on intron-containing and intronless RNAs, excluding the possibility that it is associated with the splicing machinery.

Adenosine Triphosphate↗
PubMed1996

Involvement of Escherichia coli exonuclease III and endonuclease IV in the repair of singlet oxygen-induced DNA damage.

Singlet molecular oxygen (1O2) has been implicated in several biological processes that may lead to genetic damage. The relevance of various repair pathways in plasmid inactivation mediated by 1O2 was investigated. Plasmid treated with 1O2, chemically generated, was transfected into Escherichia coli strains deficient in genes implicated in the DNA repair of oxidative damage. The ability to transform bacteria is significantly reduced in the double mutant xth,nfo, deficient in both exonuclease III and endonuclease IV, although it was similar to wild-type cells in single mutants. The products of these two genes are able to cleave DNA damaged by 1O2 and to remove DNA polymerization blocks from 3'-termini generated either directly by 1O2 treatment or after the action of the formamidopyrimidine-DNA-N-glycosylase (Fpg protein). The results indicate that the exonuclease III and endonuclease IV participate in the excision of lethal lesions induced in DNA by 1O2.

DNA Damage↗
PubMed1996

Recombination-dependent growth in exonuclease-depleted recBC sbcBC strains of Escherichia coli K-12.

Analysis of the aroLM-sbcCD interval of the Escherichia coli K-12 chromosome revealed a new gene (rdgC) encoding a function required for growth in recombination-deficient recBC sbcBC strains. Deletion of rdgC does not reduce viability, conjugational recombination, or DNA repair in rec+, recA, recB, recF, or recJ mutants. However, it makes the growth of recBC sbcBC strains reliant on the RecA, RecF, and RuvC proteins and, to a large extent, on RuvAB. The recBC sbcBC delta rdgC ruvAB construct forms colonies, but cell viability is reduced to < 5%. A recBC sbcBC delta rdgC derivative carrying the temperature-sensitive recA200 allele grows at 32 degrees but not 42 degrees. Multicopy rdgC+ plasmids reduce the growth rate of recBC sbcBC strains, while multicopy sbcC+ plasmids that reactivate SbcCD nuclease cannot be maintained without RdgC protein. The data presented are interpreted to suggest that exonuclease-depleted recBC sbcBC strains have difficulty removing the displaced arm of a collapsed replication fork and that this problem is compounded in the absence of RdgC. Recombination then becomes necessary to repair the fork and allow chromosome duplication to be completed. The possibility that RdgC is an exonuclease is discussed.

Amino Acid Sequence↗
PubMed1983

The coupled use of 'footprinting' and exonuclease III methodology for RNA polymerase binding and initiation. Application for the analysis of three tandem promoters at the control region of colicin El.

In order to determine the initiation site for three promoters P1, P2 and P3 (5' to 3') in close proximity in the colicin E1 control region we developed a new methodology that couples ternary complex formation and the analysis of the 3' border protected from exonuclease III digestion. The initiation of transcription could be detected by measuring the shift in the position of the 3' protected border when RNA polymerase moved from its binary complex position to its ternary complex position. The latter stops at a specific nucleotide because transcription is initiated with one or more NTPs missing. This approach, coupled with "footprinting", can also be used to decide whether the formation of an RNA polymerase binary or ternary complex at one site excludes or weakens binding at neighboring sites. The location of 3' protected borders reveals the formation of respective binary and ternary complexes at non-saturating RNA polymerase conditions, whereas at saturating conditions only the distal 3' boundary is seen and exonuclease cannot penetrate further. However, if "footprinting" reveals proximal 5' patterns this establishes that simultaneous binding has occurred on the same DNA fragment. The data showed that this was true for P1 and P3 which are only 8 nucleotides apart. P2 could only be detected at non-saturating conditions since it overlaps both P1 and P3. The evidence from the literature and this study establishes P1 as the true colicin E1 promoter with the possibility that supercoiling may eliminate any role for P2 and P3.

Amino Acid Sequence↗
PubMed1986

Complete nucleotide sequence of recD, the structural gene for the alpha subunit of Exonuclease V of Escherichia coli.

Intracellular amplification of the Escherichia coli RecB and RecC proteins does not result in an increase in Exonuclease V activity unless the level of a third protein, encoded between the recB and argA genes, is also amplified. Nucleotide sequence analysis of this region reveals a 1,824 nucleotide open reading frame which would encode a protein of 608 amino acids with a calculated molecular weight of 66,973. This is assumed to be the structural gene for the alpha subunit of Exonuclease V, recently designated recD. The proposed initiation codon of the recD gene overlaps the termination codon of the upstream recB gene by one nucleotide, suggesting that these genes may form an operon. The deduced amino acid sequence of the RecD protein contains a region which is homologous to highly conserved sequences in adenine nucleotide binding proteins.

Amino Acid Sequence↗
PubMed1990

Ordered deletions for DNA sequencing and in vitro mutagenesis by polymerase extension and exonuclease III gapping of circular templates.

A simple method is described for generating nested deletions from any fixed point in a cloned inset. Starting with a single-stranded phagemid template, T4 DNA polymerase is used to extend an annealed primer. This leads to a fully double-stranded circular molecule with a nick or small gap just 5' to the primer. Exonuclease III initiates progressive digestion from the resulting 3' end. Removal of timed aliquots and digestion with a single-strand specific endonuclease leads to a series of linear nested fragments having a common end corresponding to the 5' end of the primer. These molecules are circularized and used to transform cells, providing large numbers of deletion clones with targeted breakpoints. The 6-step procedure involves successive additions to tubes, beginning with a single-stranded template and ending with transformation; no extractions, precipitations or centrifugations are needed. Results are comparable to those obtained with standard Exonuclease III-generated deletion protocols, but there is no requirement for restriction endonuclease digestion or for highly purified double-stranded DNA starting material. This procedure provides a strategy for obtaining nested deletions in either direction both for DNA sequencing and for functional analysis.

Animals↗
PubMed1991

Eliminating primers from completed polymerase chain reactions with exonuclease VII.

Single-stranded oligonucleotide primers can be efficiently removed after PCR using E.coli exonuclease VII. Even only a few molecules of double stranded PCR product are unaffected by a treatment which eliminates 20 picomoles of primer in the presence of 500 ng of denatured genomic DNA. Exonuclease VII treatment is rapid and could simplify complicated multistep PCR protocols.

Electrophoresis, Agar Gel↗