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PubMed2002

A novel structure-specific endonuclease activity associated with polypyrimidine-tract binding (PTB) related protein from rat testis.

Nucleases are involved in the processing of various intermediates generated during crucial DNA metabolic processes such as replication, repair, and recombination and also during maturation of RNA precursors. An endonuclease, degrading specifically single-stranded circular DNA, was identified earlier in rat testis nuclear extract while purifying a strand-transfer activity. We are now reporting the purification of this endonuclease, which is a monomeric 42 kDa protein, from rat testis to near-homogeneity. In addition to degrading single-stranded circular DNA, it nicks supercoiled plasmid DNA to generate relaxed DNA and does not act on linear single-stranded or double-stranded DNA. It also makes specific incisions at the single-strand/duplex junction of pseudo-Y, 3'- and 5'-overhangs and 3'- and 5'-flap structures. Other structures such as mismatch, insertion loop, and Holliday junction are not substrates for the testis endonuclease. In contrast to FEN1, the testis endonuclease makes asymmetric incisions on both strands of the branched structures, and free single-stranded ends are not necessary for the structure-specific incisions. Neither 5'-3' nor 3'-5' exonuclease activity is associated with the testis endonuclease. The amino acid sequences of tryptic peptides of the 42 kDa endonuclease show near-identity to polypyrimidine-tract binding protein (PTB) that is involved in the regulation of splicing of eukaryotic mRNA. The significance of the results on the association of structure-specific endonucleae activities with PTB-related protein is discussed.

Amino Acid Sequence↗
PubMed2003

Fidelity of DNA polymerase delta holoenzyme from Saccharomyces cerevisiae: the sliding clamp proliferating cell nuclear antigen decreases its fidelity.

DNA polymerases delta and epsilon (pol delta and epsilon) are the two major replicative polymerases in the budding yeast Saccharomyces cerevisiae. The fidelity of pol delta is influenced by its 3'-5' proofreading exonuclease activity, which corrects misinsertion errors, and by enzyme cofactors. PCNA is a pol delta cofactor, called the sliding clamp, which increases the processivity of pol delta holoenzyme. This study measures the fidelity of 3'-5' exonuclease-proficient and -deficient pol delta holoenzyme using a synthetic 30mer primer/100mer template in the presence and absence of PCNA. Although PCNA increases pol delta processivity, the presence of PCNA decreased pol delta fidelity 2-7-fold. In particular, wild-type pol delta demonstrated the following nucleotide substitution efficiencies for mismatches in the absence of PCNA: G.G, 0.728 x 10(-4); T.G, 1.82 x 10(-4); A.G, <0.01 x 10(-4). In the presence of PCNA these values increased as follows: G.G, 1.30 x 10(-4); T.G, 2.62 x 10(-4); A.G, 0.074 x 10(-4). A similar but smaller effect was observed for exonuclease-deficient pol delta (i.e., 2-4-fold increase in nucleotide substitution efficiencies in the presence of PCNA). Thus, the fidelity of wild-type pol delta in the presence of PCNA is more than 2 orders of magnitude lower than the fidelity of wild-type pol epsilon holoenzyme and is comparable to the fidelity of exonuclease-deficient pol epsilon holoenzyme.

DNA Polymerase II↗
PubMed1996

Role of calf RTH-1 nuclease in removal of 5'-ribonucleotides during Okazaki fragment processing.

The role of the exonucleolytic activity of the calf 5' to 3' exo/endonuclease, a RAD2 homolog 1 (RTH-1) class nuclease, in lagging-strand DNA replication has been examined using model Okazaki fragment substrates. These substrates exemplify the situation in Okazaki fragment processing which occurs after the initiator RNA primer is cleaved off, and released intact, by calf RNase HI, leaving a single ribonucleotide at the 5' end of the RNA-DNA junction. This final RNA is then removed by the calf RTH-1 nuclease [Turchi et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9803-9807]. The cleavage specificity of calf RTH-1 nuclease for different junction ribonucleotides was compared. These were removed without the usual requirement of calf RTH-1 for an immediately adjacent upstream primer. In most cases, the presence of an upstream DNA or RNA primer, separated from the monoribonucleotide-DNA segment by either a nick or a gap, reduced the efficiency of removal of the monoribonucleotide compared to the removal seen with no upstream primer. Substrates in which the monoribonucleotide-DNA segment had been replaced by an oligomer of the same sequence but consisting entirely of DNA also exhibited upstream primer inhibition. Results with various sequences indicated that the upstream primer is generally inhibitory for ribonucleotide removal but is sometimes neutral. For deoxynucleotide removal it could be stimulatory, neutral, or inhibitory. Possible reasons for the unexpected lack of upstream primer dependence have been explored. The ratio of RNase HI to RTH-1 was also shown to be critical for both enzymes to work together efficiently. These results suggest that regions of upstream primer inhibition within the genome may play a role in determining the mechanism by which mammalian Okazaki fragments are processed.

Animals↗
PubMed1996

Kinetic mechanism of the 3'-->5' proofreading exonuclease of DNA polymerase III. Analysis by steady state and pre-steady state methods.

DNA polymerase III holoenzyme is the major replicative enzyme in Escherichia coli. An important component of the high-fidelity DNA synthesis that is characteristic of DNA polymerase III holoenzyme is the 3'-->5' proofreading exonuclease activity resident in the epsilon subunit. Steady state and pre-steady state conditions have been used to determine equilibrium and Michaelis constants for substrate binding and the rate constant for cleavage by purified epsilon subunit. The steady state kinetic constants are K(m) = 16 +/- 6 microM and kcat = 210 +/- 23 s-1 for degradation of single-stranded DNA by epsilon. These steady state values are in agreement with the rate constants determined for excision of the 3' nucleotide of a dT10 oligomer under pre-steady state conditions. Using a simple two-step model, E + Dn reversible E.Dn-->E + Dn-1, we find K = 12 microM and kf = 280 s-1 for the dT10 substrate. In these experiments, epsilon subunit acts in a distributive manner and product release is not the rate-limiting step. Activity of the epsilon subunit on paired DNA oligonucleotides with zero to three mismatches at the 3' terminus indicates that an additional step is required in the mechanism. In the scheme Dn reversible Dn* + E reversible E.Dn*-->E + Dn-1, the 3' terminus undergoes a conformational change or "melts" before the DNA is a substrate for epsilon subunit. With this additional step, the values for binding of activated substrate and cleavage are the same as those for single-stranded DNA. The kinetics for exonucleolytic degradation of single-stranded, paired, and mispaired oligonucleotides support the model that the rate-limiting step in exonucleolytic proofreading of DNA by epsilon subunit is the DNA-melting step.

Base Composition↗
PubMed1996

Interactions of the RecBCD enzyme from Escherichia coli and its subunits with DNA, elucidated from the kinetics of ATP and DNA hydrolysis with oligothymidine substrates.

Oligothymidines eight nucleotides or longer stimulate ATP hydrolysis by the RecBC and RecBCD enzymes, and they are substrates for the ATP-stimulated nuclease activity of RecBCD. The steady-state kinetics of ATP hydrolysis by the RecBC enzyme are consistent with a single ATPase and DNA binding site. Results with RecBCD and RecBCD-K177Q [an enzyme with a Lys-to-Gln mutation in the ATP binding motif of the RecD subunit [Korangy, F., & Julin, D. A. (1992) J. Biol. Chem. 267, 1727-1732]] indicate that ATP hydrolysis by the RecB subunit is stimulated by pd(T)12 binding to a high-affinity site, while the RecD subunit hydrolyzes ATP stimulated by pd(T)12 binding to a low-affinity site. The site which stimulates RecB has about 50-fold greater affinity for DNA in either RecBCD or RecBCD-K177Q than does the corresponding site in RecBC. The rates of ATP hydrolysis observed for the RecBCD enzyme at low concentrations of pd(T)12 are best explained by a mechanism where the enzyme binds to the DNA and catalyzes multiple rounds of ATP hydrolysis before dissociating. Larger DNA molecules [pd(T)25-30 and poly(dT)] are bound more tightly by RecBCD, are hydrolyzed more rapidly, and are much more effective in stimulating ATP hydrolysis than is pd(T)12. The results at low ATP concentrations where the nuclease activity is minimal (5 microM) suggest that ATP hydrolysis is stimulated by the DNA ends, but there is no evidence that the RecBCD enzyme moves along these DNA molecules in an ATP-dependent manner under these conditions.

Adenosine Triphosphatases↗
PubMed1997

Evidence for a recombination-independent pathway for the repair of DNA interstrand cross-links based on a site-specific study with nitrogen mustard.

DNA-DNA interstrand cross-links are thought to be important for the cytotoxicity of many chemotherapeutic agents. To study this more definitively, adduct site-specific methods are used to construct a plasmid with a single nitrogen mustard interstrand cross-link (inter-HN2-pTZSV28). Replication efficiency (RE = [colonies from (inter-HN2-pTZSV28)/(control with no cross-link)]) is approximately 0.3 following transformation into Escherichia coli, implying that the cross-link is repaired. The commonly accepted pathway for cross-link repair, which involves both nucleotide excision repair (NER) and recombination, is ruled out since RE is approximately 0.3 in a delta recA strain. Non-RecA-directed recombination such as copy-choice is also unlikely. However, NER is involved since RE was approximately 0.02 in strains deficient in NER. Base excision repair is not important since RE is approximately 0.3 in strains deficient in 3-methyladenine DNA glycosylases I and II, FAPY DNA glycosylase, both known apurinic/apyrimidinic endonucleases, or DNA deoxyribophosphodiesterase. Another hypothetical repair pathway hinging on a 5' --> 3' exonuclease activity is unlikely since RE is approximately 0.3 in cells deficient in either the 5' --> 3' exonuclease activities of DNA polymerase I, exonuclease VII, or RecJ. Thus, aside from NER, it is unclear what else participates in this recombination-independent repair pathway, although a pathway involing NER followed by replicative bypass of the lesion is the current working hypothesis. Psoralen interstrand cross-links appear not to be repairable by this second pathway, which may have implications for the relative cytotoxicity of interstrand cross-links from different agents.

DNA Adducts↗
PubMed1997

Purification and characterization of the DNA polymerase alpha associated exonuclease: the RTH1 gene product.

We report here the purification and mechanistic characterization of a 5'-3' exonuclease associated with DNA polymerase alpha from the yeast Saccharomyces cerevisiae. Earlier, we identified a 5' --> 3' exonuclease activity that copurified with yeast DNA polymerase alpha-primase in a multiprotein complex [Biswas, E. E., et al. (1993) Biochemistry, 32, 3020-3027]. Peptide sequence analysis of the purified 47 kDa exonuclease was carried out, and the peptide sequence was found to be identical to the S. cerevisiae gene YKL510 encoded polypeptide, which is also known as yeast RAD2 homolog 1 or RTH1 nuclease. The native exonuclease also had strong flap endonuclease activity similar to that observed with RTH1 nuclease and homologous yeast (RAD2) and mammalian enzymes. During our studies, we have discovered certain unique features of the mechanism of action of the native RTH1 nuclease. Studies presented here indicated that the exonuclease had specific pause sites during its 5'-3' exonuclease nucleotide excision. These pause sites were easily detected with long (approximately 50 bp) oligonucleotide substrates during exonucleolytic excision by the formation of a discontinuous ladder of excision product. We have further analyzed the mechanism of generation of the pause sites, as they could occur through a number of different pathways. Alignment of the pause sites with the nucleotide sequence of the oligonucleotide substrate indicated that the pause sites were dependent on the nucleotide sequence. Our analysis revealed that RTH1 nuclease pauses predominantly at G:C rich sequences. With poly(dA):oligo(dT)50 as substrate, the exonucleolytic products formed a continuous ladder with no evidence of pausing. The G:C rich DNA sequences are thermodynamically more stable than the A:T rich sequences, which may be in part responsible for pausing of the RTH1 5' --> 3' exonuclease at these sites.

Amino Acid Sequence↗
PubMed1997

Stimulation of RTH1 nuclease of the yeast Saccharomyces cerevisiae by replication protein A.

The RTH1 nuclease is involved in the replication of chromosomal DNA as well as in the repair of DNA damage. Replication protein A (RPA) is also an integral part of the DNA replication and repair processes. We have investigated the roles(s) of RPA in the function of RTH1 nuclease, including its structure specific endonuclease activity. Initial in vitro studies, which employed a "flap" or a "pseudo Y" substrate containing a short 14 bp duplex region, showed the effect of RPA to be minimal or inhibitory. As RPA inhibition is unwarranted for a protein participating in the DNA replication process, we have further investigated the mechanism of such inhibition. Alternate flap and pseudo Y substrates with a long duplex region (50 bp) were prepared using M13mp19 ssDNA and synthetic oligonucleotides. Yeast RPA stimulated the endonuclease activity of RTH1 endonuclease with these substrates in a dose-dependent manner. Kinetic analysis suggested that yRPA exerted a bipartite effect on the nuclease reaction: (i) the "load time" of RTH1 nuclease onto the DNA substrate decreased from approximately 5 to 2 min in the presence of RPA, and (ii) following initiation of the nuclease reaction, the initial rate of the reaction increased 10-fold in the presence of yRPA. Further analysis of the interaction of RPA with various endonuclease substrates indicated that RPA has a weak helix destabilizing effect and could melt small, 14 bp, regions of duplex DNA. RTH1 endonuclease cleaves the DNA strand at the junction of single- and double-stranded DNA; consequently, the observed inhibition with small duplex substrates was likely due to duplex melting. Our studies also demonstrated that RPA stimulated the RNase H activity of RTH1 nuclease significantly. In both instances (RTH1 endonuclease and RNase H), the stimulation may involve a specific interaction of RPA with the RTH1 nuclease rather than a structural positioning of the DNA substrate by RPA.

Bacteriophage M13↗
PubMed1997

Inhibition of klenow fragment (exo-) catalyzed DNA polymerization by (5R)-5,6-dihydro-5-hydroxythymidine and structural analogue 5,6-dihydro-5-methylthymidine.

Oligonucleotides containing 5R-5,6-dihydro-5-hydroxythymidine (5R-3) and structural analogue 5,6-dihydro-5-methylthymidine (9) at defined sites were chemically synthesized via a method that obviates the use of NH4OH. Oligonucleotides prepared by this method were used to examine the effects of 5R-3 and 9 on the fidelity of Klenow (exo-) in vitro. The presence of lesions 5R-3 and 9 in DNA templates was shown to inhibit polymerization of primers hybridized to these templates. Inhibition was observed for both translesional synthesis and extension one nucleotide past the lesion, with the latter being more pronounced. The fidelity of Klenow (exo-) was reduced only slightly when utilizing substrates containing either dihyropyrimidine nucleotide. These results provide the first experimental verification of computational studies carried out on the effects of 3 on DNA templates, and are consistent with a structural model in which the C5-methyl group of 5R-3 adopts a pseudoaxial orientation resulting in a disruption in base stacking.

DNA↗
PubMed1998

Recognition of sequence-directed DNA structure by the Klenow fragment of DNA polymerase I.

Time-resolved fluorescence spectroscopy was used to investigate the influence of sequence-directed DNA structure upon the interaction between the Klenow fragment of DNA polymerase I and a series of defined oligonucleotide primer/templates. 17/27-mer (primer/template) oligonucleotides containing a dansyl fluorophore conjugated to a modified deoxyuridine residue within the primer strand were used as substrates for binding to Klenow fragment. The time-resolved fluorescence anisotropy decay of the dansyl probe was analyzed in terms of two local environments, either solvent-exposed or buried, corresponding to primer/templates positioned with the primer 3' terminus in the polymerase site or the 3'-5' exonuclease site of the enzyme, respectively. Equilibrium constants for partitioning of DNA between the two sites were evaluated from the anisotropy decay data for primer/templates having different (A + T)-rich sequences flanking the primer 3' terminus. Primer/templates with AAAATG/TTTTAC and CGATAT/GCTATA terminal sequences (the nucleotides on the left refer to the last six bases at the 3' end of the primer, and the nucleotides on the right are the corresponding bases in the template) were bound mostly at the polymerase site. The introduction of single mismatches opposite the primer 3' terminus of these DNA substrates increased their partitioning into the 3'-5' exonuclease site, in accord with the results of an earlier study [Carver, T.E., Hochstrasser, R.A., and Millar, D.P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10670-10674]. In contrast, a primer/template with the terminal sequence CAATTT/GTTAAA, containing an A-tract element AATTT, exhibited a surprising preference for binding at the 3'-5' exonuclease site, despite the absence of mismatched bases in the DNA substrate. Interruption of the A-tract with a single AG step, to give the terminal sequence CAGTTT/GTCAAA, reversed the effect of the A-tract, causing the DNA to partition in favor of the polymerase site. Moreover, the presence of a single mismatch opposite the primer 3' terminus was also sufficient to reverse the effect of the A-tract, resulting in a distribution of DNA between polymerase and 3'-5' exonuclease sites that was similar to that observed for the other mismatched DNA substrates. Taken together, these results suggest that the A-tract adopts an unusual conformation that is disruptive to binding at the polymerase site. The effect of the A-tract on binding of DNA to the polymerase site is discussed in terms of the unusual helix structural parameters associated with these sequence elements and the difference between the local geometry of the A-tract and the conformation adopted by duplex DNA within the polymerase cleft. The results of this study show that in addition to base mismatches, Klenow fragment can also recognize irregularities in the helix geometry of perfectly base-paired DNA.

Base Composition↗
PubMed1998

Amino acid residues involved in determining the processivity of the 3'-5' exonuclease activity in a family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus.

Herein, we report on the mutational analysis of a 70-amino acid segment (region 1, residues 438-508) of family B DNA polymerase from the thermoacidophilic archaeon Sulfolobus solfataricus (Sso DNA pol). Region 1, which lies between the Exo III sequence and the similarity motif D- -SLYP, connects the exonuclease and polymerase domains of Sso DNA pol. Two C-terminally deleted forms of the enzyme, proteins N438 (residues 1-438) and N508 (residues 1-508), were overproduced in the recombinant form and biochemically characterized. They contain the three evolutionarily conserved Exo motifs, but differ in the extent of the C-terminal deletion, since only N508 includes region 1. Both have been found to retain a Mn2+-dependent 3'-5' exonuclease activity, whose thermal stability appears to be increased in comparison to that of the full-sized enzyme. Assays for processive 3'-5' exonuclease activity, carried out with the heparin trap method on a 24-base oligonucleotide, have revealed that protein N508, as well as the full-length Sso DNA pol, retains a level of processivity of the degradative function substantially higher than that for protein N438. In addition, six site-specific mutations have been introduced at the highly conserved Y-GG/A motif, which has been found within Sso DNA pol region 1. All mutant proteins (Lys491Ile, Tyr495Ser, Lys496Ile, Gly497Ala, and Ala498Val) display increased processivity of their 3'-5' exonuclease activity, with the exception of protein Tyr495Phe. By a steady-state kinetic analysis of the exonucleolytic reaction on a 24-base oligonucleotide, the above site-specific mutations have been found to affect Km values consistently with the observed differences in the processivity values, whereas the effect on the kcat values seems to be less important. The results from this mutational analysis indicate that region 1 is involved in determining the processivity of the proofreading function, directly interacting with the nucleic acid substrate.

Amino Acids↗
PubMed1999

Structures of normal single-stranded DNA and deoxyribo-3'-S-phosphorothiolates bound to the 3'-5' exonucleolytic active site of DNA polymerase I from Escherichia coli.

The interaction of a divalent metal ion with a leaving 3' oxygen is a central component of several proposed mechanisms of phosphoryl transfer. In support of this are recent kinetic studies showing that thiophilic metal ions (e.g., Mn2+) stimulate the hydrolysis of compounds in which sulfur takes the place of the leaving oxygen. To examine the structural basis of this phenomenon, we have solved four crystal structures of single-stranded DNA's containing either oxygen or sulfur at a 3'-bridging position bound in conjunction with various metal ions at the 3'-5' exonucleolytic active site of the Klenow fragment (KF) of DNA polymerase I from Escherichia coli. Two structures of normal ssDNA bound to KF in the presence of Zn2+ and Mn2+ or Zn2+ alone were refined at 2.6- and 2.25-A resolution, respectively. They serve as standards for comparison with other Mn2+- and Zn2+-containing structures. In these cases, Mn2+ and Zn2+ bind at metal ion site B in a nearly identical position to Mg2+ (Brautigam and Steitz (1998) J. Mol. Biol. 277, 363-377). Two structures of KF bound to a deoxyoligonucleotide that contained a 3'-bridging sulfur at the scissile phosphate were refined at 2.03-A resolution. Although the bridging sulfur compounds bind in a manner very similar to that of the normal oligonucleotides, the presence of the sulfur changes the metal ion binding properties of the active site such that Mn2+ and Zn2+ are observed at metal ion site B, but Mg2+ is not. It therefore appears that the ability of the bridging sulfur compounds to exclude nonthiophilic metal ions from metal ion site B explains the low activity of KF exonuclease on these substrates in the presence of Mg2+ (Curley et al. (1997) J. Am. Chem. Soc. 119, 12691-12692) and that the 3'-bridging atom of the substrate is influencing the binding of metal ion B prior to catalysis.

Binding Sites↗
PubMed1999

Interaction of DNA polymerase I (Klenow fragment) with DNA substrates containing extrahelical bases: implications for proofreading of frameshift errors during DNA synthesis.

Frameshift mutagenesis occurs through the misalignment of primer and template strands during DNA synthesis and involves DNA intermediates that contain one or more extrahelical bases in either strand of the DNA substrate. To investigate whether these DNA structures are recognized by the proofreading apparatus of DNA polymerases, time-resolved fluorescence spectroscopy was used to examine the interaction between the Klenow fragment of DNA polymerase I and synthetic DNA primer-templates containing extrahelical bases at defined positions within the template strand. A dansyl probe attached to the DNA was used to measure the fractional occupancies of the polymerase and 3'-5' exonuclease sites of the enzyme for DNA substrates with and without the extrahelical bases. The presence of an extrahelical base at the first position from the primer 3' terminus increased the level of partitioning of the DNA substrates into the 3'-5' exonuclease site by 3-7-fold, relative to the perfectly base-paired primer-template, depending on the identity of the extrahelical base. The ability of different extrahelical bases to promote partitioning of DNA into the 3'-5' exonuclease site decreased in the following order: G > A approximately T > C. The results of partitioning measurements for DNA substrates containing a bulged adenine base at different positions within the template showed that an extrahelical base is recognized up to five bases from the primer 3' terminus. The largest effects were observed for the extrahelical base at the third or fourth positions from the primer terminus, which increased the level of partitioning of DNA into the 3'-5' exonuclease site by 8- and 18-fold, respectively, relative to that of the perfectly base-paired substrate. Steady-state fluorescence measurements of analogous primer-templates containing 2-aminopurine (AP) at the primer 3' terminus indicate that extrahelical bases increase the degree of terminus unwinding, especially when close to the terminus. In addition, steady-state kinetic measurements of removal of AP from the primer-templates indicate that the exonucleolytic cleavage activity of Klenow fragment is correlated with the increased level of partitioning of bulged DNA substrates to the 3'-5' exonuclease site relative to that of properly base-paired DNA. The results of this study indicate that misalignment of primer and template strands to generate an extrahelical base strongly promotes transfer of a DNA substrate to the 3'-5' exonuclease site, suggesting that the premutational intermediates in frameshift mutagenesis are subject to proofreading by the polymerase.

2-Aminopurine↗
PubMed1999

Effect of flap modifications on human FEN1 cleavage.

The flap endonuclease, FEN1, plays a critical role in DNA replication and repair. Human FEN1 exhibits both a 5' to 3' exonucleolytic and a structure-specific endonucleolytic activity. On primer-template substrates containing an unannealed 5'-tail, or flap structure, FEN1 employs a unique mechanism to cleave at the point of annealing, releasing the 5'-tail intact. FEN1 appears to track along the full length of the flap from the 5'-end to the point of cleavage. Substrates containing structural modifications to the flap have been used to explore the mechanism of tracking. To determine whether the nuclease must recognize a succession of nucleotides on the flap, chemical linkers were used to replace an interior nucleotide. The nuclease could readily traverse this site. The footprint of the nuclease at the time of cleavage does not extend beyond 25 nucleotides on the flap. Eleven-nucleotide branches attached to the flap beyond the footprinted region do not prevent cleavage. Single- or double-thymine dimers also allow cleavage. cis-Platinum adducts outside the protected region are moderately inhibitory. Platinum-modified branch structures are completely inert to cleavage. These results show that some flap modifications can prevent or inhibit tracking, but the tracking mechanism tolerates a variety of flap modifications. FEN1 has a flexible loop structure through which the flap has been proposed to thread. However, efficient cleavage of branched structures is inconsistent with threading the flap through a hole in the protein.

Base Sequence↗
PubMed1999

DNA hydrolytic activity associated with the Ustilago maydis REC1 gene product analyzed on hairpin oligonucleotide substrates.

The REC1 gene of Ustilago maydis functions in the maintenance of genome stability as evidenced by the mutator phenotype resulting from inactivation of the gene. The biochemical function of the Rec1 protein was previously identified as a 3'-5'-directed DNA exonuclease. Here studies on the mechanism of action of Rec1 were performed using radiolabeled oligonucleotide DNAs as substrates, enabling detection of single cleavage events after electrophoresis on DNA sequencing gels. The oligonucleotides that were utilized were designed to be self-annealing so that they formed hairpin structures. This simplified interpretation of the data since each molecule contained only one 3'-terminus. Analysis revealed that digestion proceeded by a distributive mode of action and that degradation of DNA was governed by an interplay between sequence context and conformation. The preferential substrate was DNA with a recessed 3'-end. It was discovered that the enzyme had abasic endonuclease activity, was capable of initiating at an internal nick, and had no preference for mismatched bases either internally or terminally. Endonucleolytic cleavage was 5' to the abasic site.

Apurinic Acid↗
PubMed2000

In vitro reconstitution of the Schizosaccharomyces pombe alternative excision repair pathway.

Schizosaccharomyces pombe alternative excision repair has been shown genetically and biochemically to be involved in the repair of a wide variety of DNA lesions. AER is initiated by a damage-specific endonuclease (Uve1p) that recognizes UV-induced photoproducts, base mispairs, abasic sites, and platinum G-G diadducts and cleaves the DNA phosphodiester backbone 5' to a lesion. Several models exist that employ various mechanisms for damage removal based on the activities of Rad2p, a nuclease thought to be responsible for damage excision in AER. This study represents the first report of the biochemical reconstitution of the AER pathway. A base mispair-containing substrate is repaired in a reaction requiring S. pombe Uve1p, Rad2p, DNA polymerase delta, replication factor C, proliferating cell nuclear antigen, and T4 DNA ligase. Surprisingly, damage is removed exclusively by the 5' to 3' exonuclease activity of Rad2p and not its "flap endonuclease" activity and is absolutely dependent upon the presence of the 5'-phosphoryl moiety at the Uve1p cleavage site.

DNA Damage↗
PubMed2001

The "flap" endonuclease gene FEN1 is excluded as a candidate gene implicated in the CAG repeat expansion underlying Huntington disease.

At least 12 disorders including Huntington disease (HD) are associated with expansion of a trinucleotide repeat (TNR). Factors contributing to the risk of expansion of TNRs and the mechanism of expansion have not been elucidated. Data from Saccharomyces cerevisiae suggest that the flap endonuclease FEN1 plays a role in expansion of repetitive DNA tracts. It has been hypothesized that insufficiency of FEN1 or a mutant FEN1 might contribute to the occurrence of expansion events of long repetitive DNA tracts after polymerase slippage events during lagging strand synthesis. The expression pattern of FEN1 was determined, and ubiquitous tissue expression, including germ cells, suggested that FEN1 has the potential to be involved in HD. Fifteen HD parent/child pairs that demonstrated intergenerational increases in CAG length of greater than 10 repeats were examined for possible mutations or polymorphisms within the FEN1 gene that could underlie the saltatory repeat expansions seen in these individuals. No alterations were observed compared to 50 controls, excluding FEN1 as a trans-acting factor underlying TNR expansion. The identification of a candidate gene(s) in HD or other CAG-expansion disorders implicated in TNR instability will elucidate the mechanism of expansion for this growing family of neurological disorders.

Animals↗