The Experts below are selected from a list of 174 Experts worldwide ranked by ideXlab platform
Aziz Sancar - One of the best experts on this subject based on the ideXlab platform.
-
INDIRECT ROLE IN 5 ’ INCISION*
2015Co-Authors: Jing-jer Lin, John E Hearst, Meleah A. Phillips, Aziz SancarAbstract:UvrB plays a central role in (A)BC Excinuclease. To study its role in the incision reactions, conserved His and Asp residues in this subunit were mutagenized. All His and the majority of Asp mutants behaved like wild-type protein in vivo and in vitro. However, three mu-tants, D337A, D478A, and D510A, either completely or partially abolished UvrB activity. All three mutant proteins associate with UvrA normally but D337A and D510A were unable to bind to DNA specifically. The UvrB-D478A mutant bound to DNA specifically but failed to denature and kink the DNA. However, UvrB-D478A was efficiently loaded onto DNA preincised at the 3 ‘ site and promoted near-normal incision by UvrC at the 5 ’ site. We propose that D478 is involved in bending DNA and catalysis of the 3 ‘ incision and that the 3 ’ incision precedes the 5 ’ incision. UvrB ’ which is missing the carboxyl-terminal 43 amino acids binds to, and kinks DNA but is unable to make the 3 ’ incision suggesting that it is missing a residue involved in ca-talysis. This residue was identified to be E639 by site-specific mutagenesis. The UvrB subunit of (A)BC Excinuclease plays a central role in the excision reaction because it interacts with all of the components involved in excision repair, including UvrA
-
reconstitution of human dna repair excision nuclease in a highly defined system
Journal of Biological Chemistry, 1995Co-Authors: David Mu, Chi Hyun Park, Tsukasa Matsunaga, Joyce T Reardon, Aziz SancarAbstract:Abstract Xeroderma pigmentosum is a hereditary disease caused by defective DNA repair. Somatic cell genetics and biochemical studies with cell-free extracts indicate that at least 16 polypeptides are required to carry out the repair reaction proper, i.e. the removal of the lesion from the DNA by the dual incisions of the damaged strand. To find out if these proteins are necessary and sufficient for excision repair, they were obtained at a high level of purity in five fractions. The mixture of these five fractions reconstituted the excision nuclease (Excinuclease) activity. Using the reconstituted Excinuclease, we found that the excised fragment remains associated with the post-incision DNA-protein complex, suggesting that accessory proteins are needed to release the excised oligomer.
-
substrate spectrum of human Excinuclease repair of abasic sites methylated bases mismatches and bulky adducts
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Juch Chin Huang, David S Hsu, Aleksey Kazantsev, Aziz SancarAbstract:Nucleotide-excision repair is the repair system for removing bulky lesions from DNA. Humans deficient in this repair pathway suffer from xeroderma pigmentosum (XP), a disease characterized by photodermatoses, including skin cancers. At the cellular level, XP patients fail to remove cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts induced by UV light, as well as other bulky DNA lesions caused by various genotoxic agents. XP cells are not particularly sensitive to ionizing radiation or to alkylating agents that cause mostly nonbulky DNA lesions. Therefore, it has generally been assumed that the human nucleotide-excision repair enzyme (Excinuclease) is specific for bulky adducts. To determine the substrate range of human Excinuclease we used the highly sensitive excision assay and tested bulky adducts, synthetic apurinic/apyrimidinic sites, N6-methyladenine, O6-methylguanine, and mismatches as potential substrates. We found that all of these "lesions" were removed by human Excinuclease, although with vastly different efficiencies.
-
determination of minimum substrate size for human Excinuclease
Journal of Biological Chemistry, 1994Co-Authors: Juch Chin Huang, Aziz SancarAbstract:Human cells remove bulky adducts from DNA by excising single-stranded fragments 27-29 nucleotides in length by an enzyme system consisting of at least 14 polypeptides. All of the previous work on characterizing the excision reaction was conducted with plasmids 3 or 8 kilobases in length. To determine if the size and tertiary structure of DNA play a role in the excision reaction and to find out if large DNA fragments are necessary to contact all of the subunits of the Excinuclease, we performed experiments with circular DNA and with linear DNA fragments of various sizes. We found that the human Excinuclease is capable of removing DNA adducts from linear and covalently closed circular DNAs with about the same efficiency. Furthermore, we found that the Excinuclease can remove a thymine dimer or a psoralen-thymine monoadduct from linear fragments provided that the distance between the lesion and the 5'-terminus of the damaged strand is > or = 60 nucleotides and the distance between the lesion and the 3'-terminus is > or = 44 nucleotides. Thus, the minimum size substrate for human Excinuclease is approximately 100 base pairs in length.
-
effect of sequence adduct type and opposing lesions on the binding and repair of ultraviolet photodamage by dna photolyase and a bc Excinuclease
Journal of Biological Chemistry, 1993Co-Authors: Daniel L Svoboda, Colin A Smith, Johnstephen Taylor, Aziz SancarAbstract:The cis,syn and (6-4) products of dipyrimidine sites are the major mutagenic and lethal UV photoproducts in DNA. To investigate their relative susceptibilities to repair and other factors such as sequence context and lesions in the complementary strand that might influence repair efficiencies, we constructed 49-mer duplexes containing site-specific photoproducts of thymidylyl(3',5')thymidine sites at central locations. Using these substrates, we measured the binding of Escherichia coli DNA photolyase to cis,syn dimers in four sequence contexts and to two cis,syn dimers in close proximity and on opposing strands. We found that the sequence within a 10-base pair region had little effect on binding and that two enzyme molecules bound to substrate containing two dimers in the 5'-staggered orientation, but not in the 3'-staggered one. Similarly, the excision of a cis,syn dimer by (A)BC Excinuclease was not influenced by the sequence in the immediate vicinity of the dimer, and the enzyme was active on 5'-staggered cis,syn dimers, but not on 3'-staggered ones. Of special significance, we found that (A)BC Excinuclease removed the cis,syn, trans,syn-I, (6-4), and Dewar photoproducts at vastly differing relative rates of 1:6:9:9, respectively.
Jing-jer Lin - One of the best experts on this subject based on the ideXlab platform.
-
INDIRECT ROLE IN 5 ’ INCISION*
2015Co-Authors: Jing-jer Lin, John E Hearst, Meleah A. Phillips, Aziz SancarAbstract:UvrB plays a central role in (A)BC Excinuclease. To study its role in the incision reactions, conserved His and Asp residues in this subunit were mutagenized. All His and the majority of Asp mutants behaved like wild-type protein in vivo and in vitro. However, three mu-tants, D337A, D478A, and D510A, either completely or partially abolished UvrB activity. All three mutant proteins associate with UvrA normally but D337A and D510A were unable to bind to DNA specifically. The UvrB-D478A mutant bound to DNA specifically but failed to denature and kink the DNA. However, UvrB-D478A was efficiently loaded onto DNA preincised at the 3 ‘ site and promoted near-normal incision by UvrC at the 5 ’ site. We propose that D478 is involved in bending DNA and catalysis of the 3 ‘ incision and that the 3 ’ incision precedes the 5 ’ incision. UvrB ’ which is missing the carboxyl-terminal 43 amino acids binds to, and kinks DNA but is unable to make the 3 ’ incision suggesting that it is missing a residue involved in ca-talysis. This residue was identified to be E639 by site-specific mutagenesis. The UvrB subunit of (A)BC Excinuclease plays a central role in the excision reaction because it interacts with all of the components involved in excision repair, including UvrA
-
active site of a bc Excinuclease i evidence for 5 incision by uvrc through a catalytic site involving asp399 asp438 asp466 and his538 residues
Journal of Biological Chemistry, 1992Co-Authors: Jing-jer Lin, Aziz SancarAbstract:(A)BC Excinuclease of Escherichia coli removes damaged nucleotides from DNA by hydrolyzing the 8th phosphodiester bond 5' and the 15th phosphodiester bond 3' to the modified base. The activity results from the ordered action of UvrA, UvrB, and UvrC proteins. The role of UvrA is to help assemble the UvrB.DNA complex, and it is not involved in the actual incision reactions which are carried out by UvrB and UvrC. To investigate the role of UvrC in the nuclease activity a subset of His, Asp, and Glu residues in the C-terminal half of the protein were mutagenized in vitro. The effect of these mutations on UV resistance in vivo and incision activity in vitro were investigated. Mutations, H538F, D399A, D438A, and D466A conferred extreme UV sensitivity. Enzyme reconstituted with these mutant proteins carried out normal 3' incision but was completely defective in 5' incision activity. Our data suggest that UvrC makes the 5' incision by employing a mechanism whereby the three carboxylates acting in concert with H538 and a Mg2+ ion facilitate nucleophilic attack by an active site water molecule.
-
active site of a bc Excinuclease ii binding bending and catalysis mutants of uvrb reveal a direct role in 3 and an indirect role in 5 incision
Journal of Biological Chemistry, 1992Co-Authors: Jing-jer Lin, John E Hearst, A M Phillips, Aziz SancarAbstract:UvrB plays a central role in (A)BC Excinuclease. To study its role in the incision reactions, conserved His and Asp residues in this subunit were mutagenized. All His and the majority of Asp mutants behaved like wild-type protein in vivo and in vitro. However, three mutants, D337A, D478A, and D510A, either completely or partially abolished UvrB activity. All three mutant proteins associate with UvrA normally but D337A and D510A were unable to bind to DNA specifically. The UvrB-D478A mutant bound to DNA specifically but failed to denature and kink the DNA. However, UvrB-D478A was efficiently loaded onto DNA preincised at the 3' site and promoted near-normal incision by UvrC at the 5' site. We propose that D478 is involved in bending DNA and catalysis of the 3' incision and that the 3' incision precedes the 5' incision. UvrB which is missing the carboxyl-terminal 43 amino acids binds to, and kinks DNA but is unable to make the 3' incision suggesting that it is missing a residue involved in catalysis. This residue was identified to be E639 by site-specific mutagenesis.
-
a bc Excinuclease the escherichia coli nucleotide excision repair enzyme
Molecular Microbiology, 1992Co-Authors: Jing-jer Lin, Aziz SancarAbstract:Nucleotide excision repair is the major pathway for removing damage from DNA. (A)BC Excinuclease is the nuclease activity which initiates nucleotide excision repair in Escherichia coli. In this review, we focus on current understanding of the structure-function of the enzyme and the reaction mechanism of the repair pathway. In addition, recent biochemical studies on preferential repair of actively transcribed genes in E. coli are summarized.
-
The C-terminal half of UvrC protein is sufficient to reconstitute (A)BC Excinuclease.
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: Jing-jer Lin, Aziz SancarAbstract:Abstract The UvrC protein is one of three subunits of the Escherichia coli repair enzyme (A)BC Excinuclease. This subunit is thought to have at least one of the active sites for nucleophilic attack on the phosphodiester bonds of damaged DNA. To localize the active site, mutant UvrC proteins were constructed by linker-scanning and deletion mutagenesis. In vivo studies revealed that the C-terminal 314 amino acids of the 610-amino acid UvrC protein were sufficient to confer UV resistance to cells lacking the uvrC gene. The portion of the uvrC gene encoding the C-terminal half of the protein was fused to the 3' end of the E. coli malE gene (which encodes maltose binding protein), and the fusion protein MBP-C314C was purified and characterized. The fusion protein, in combination with UvrA and UvrB subunits, reconstituted the Excinuclease activity that incised the eighth phosphodiester bond 5' and the fourth phosphodiester bond 3' to a psoralen-thymine adduct. These results suggest that the C-terminal 314 amino acids of UvrC constitute a functional domain capable of interacting with the UvrB-damaged DNA complex and of inducing the two phosphodiester bond incisions characteristic of (A)BC Excinuclease.
Bengt Norden - One of the best experts on this subject based on the ideXlab platform.
-
structure of uvrabc Excinuclease uv damaged dna complexes studied by flow linear dichroism dna curved by uvrb and uvrc
FEBS Letters, 1992Co-Authors: Masayuki Takahashi, Elisabeth Bertrandburggraf, Robert P P Fuchs, Bengt NordenAbstract:The interaction between UvrABC Excinuclease from Escherichia coli and ultraviolet light- (UV) damaged DNA was studied by flow linear dichroism. The dichroism signal from DNA was drastically decreased in intensity upon incubation with UvrA and UvrB or whole enzyme in the presence of effector ATP. The change was specific for UV-damaged DNA, and a concluded suppressed DNA orientation suggests the wrapping of DNA around the protein. The incubation with the UvrC subunit alone also somewhat reduces the signal, however, in this case the change was smaller and not specific for UV-damaged DNA. The structural modification of DNA, promoted by the (UvrA2-UvrB) complex, probably facilitates or stabilizes the interaction of the UvrC subunit with DNA for the excision.
Elisabeth Bertrandburggraf - One of the best experts on this subject based on the ideXlab platform.
-
binding and incision activities of uvrabc Excinuclease on slipped dna intermediates that generate frameshift mutations
Journal of Molecular Biology, 1996Co-Authors: Emmanuelle Delagoutte, Elisabeth Bertrandburggraf, Iain B Lambert, Robert P P FuchsAbstract:Abstract Previous in vivo studies involving sequence 5′-CCCG 1 G 2 G 3 -3′ ( Sma I site) have demonstrated that adducts of N -2-acetylaminofluorene (AAF) to any of the three guanine residues of the Sma I sequence induce, with different efficiencies, two classes of −1 frameshift events, namely −G and −C mutations, referred to as targeted and semitargeted mutations, respectively. It has been proposed that both events occur during replication as a consequence of slippage events involving slipped mutagenic intermediates (SMIs). In order to evaluate the potential role of the UvrABC Excinuclease in frameshift mutagenesis, we have studied the interaction of this enzyme with DNA molecules mimicking SMIs in vitro . In all of our constructions, when present, the AAF adduct was located on the third guanine residue of the Sma I site (5′-CCCG 1 G 2 G 3 -3′). This strand was referred to as the top strand, the complementary strand being the bottom strand. Double-stranded heteroduplexes mimicking the targeted and semitargeted SMIs contained a deletion of a C and a G within the Sma I sequence in the bottom strand and were designated ΔC/3 and ΔG/3 when modified with the AAF on the third guanine residue in the top strand or ΔC/O and ΔG/O when unmodified. The modified homoduplex was designated Sma I/3. ΔC/O and ΔG/O were weakly recognized by UvrA 2 B, but not incised. All three AAF-modified substrates were recognized with similar efficiency and much more efficiently than unmodified heteroduplexes. With AAF-monomodified substrates, dissociation of UvrA 2 from the UvrA 2 B- DNA complex occurred more readily in heteroduplexes than in the homoduplex. Sma I/3 and ΔC/3 were incised with equal efficiency, while ΔG/3 was less incised. The position of the AAF lesion dictated the position of the incised phosphodiester bonds, suggesting that the presence of a bulge can modulate the yield but not the incision pattern of AAF-modified substrates. The finding that UvrABC Excinuclease acts on substrates that mimic SMIs suggests that the nucleotide excision repair pathway may help in fixing frameshift mutations before the following round of replication.
-
structure of uvrabc Excinuclease uv damaged dna complexes studied by flow linear dichroism dna curved by uvrb and uvrc
FEBS Letters, 1992Co-Authors: Masayuki Takahashi, Elisabeth Bertrandburggraf, Robert P P Fuchs, Bengt NordenAbstract:The interaction between UvrABC Excinuclease from Escherichia coli and ultraviolet light- (UV) damaged DNA was studied by flow linear dichroism. The dichroism signal from DNA was drastically decreased in intensity upon incubation with UvrA and UvrB or whole enzyme in the presence of effector ATP. The change was specific for UV-damaged DNA, and a concluded suppressed DNA orientation suggests the wrapping of DNA around the protein. The incubation with the UvrC subunit alone also somewhat reduces the signal, however, in this case the change was smaller and not specific for UV-damaged DNA. The structural modification of DNA, promoted by the (UvrA2-UvrB) complex, probably facilitates or stabilizes the interaction of the UvrC subunit with DNA for the excision.
-
identification of the different intermediates in the interaction of a bc Excinuclease with its substrates by dnase i footprinting on two uniquely modified oligonucleotides
Journal of Molecular Biology, 1991Co-Authors: Elisabeth Bertrandburggraf, John E Hearst, Christopher P Selby, Aziz SancarAbstract:Abstract (A)BC Excinuclease is the enzymatic activity resulting from the joint actions of UvrA, UvrB and UvrC proteins of Escherichia coli . The enzyme removes from DNA many types of adducts of dissimilar structures with different efficiencies. To understand the mechanism of substrate recognition and the basis of enzyme specificity, we investigated the interactions of the three subunits with two synthetic substrates, one containing a psoralen-thymine monoadduct and the other a thymine dimer. Using DNase I as a probe, we found that UvrA makes a 33 base-pair footprint around the psoralen-thymine adduct and that UvrA-UvrB make a 45 base-pair asymmetric footprint characterized by a hypersensitive site 11 nucleotides 5′ to the adduct and protection mostly on the 3′ side of the damage. Conditions that favor dissociation of UvrA from the UvrA-UvrB-DNA complex, such as addition of excess undamaged DNA to the reaction mixture, resulted in the formation of a 19 base-pair UvrB footprint. In contrast, a thymine dimer in a similar sequence context failed to elicit a UvrA, a UvrA-UvrB or UvrB footprint and gave rise to a relatively weak DNase I hypersensitive site typical of a UvrA-UvrB complex. Dissociation of UvrA from the UvrA-UvrB-DNA complex stimulated the rate of incision of both substrates upon addition of UvrC, leading us to conclude that UvrA is not a part of the incision complex and that it actually interferes with incision. The extent of incision of the two substrates upon addition of UvrC (70% for the psoralen adduct and 20% for the thymine dimer) was proportional to the extent of formation of the UvrA-UvrB-DNA (i.e. UvrB-DNA) complex, indicating that substrate discrimination occurs at the preincision step.
Robert P P Fuchs - One of the best experts on this subject based on the ideXlab platform.
-
binding and incision activities of uvrabc Excinuclease on slipped dna intermediates that generate frameshift mutations
Journal of Molecular Biology, 1996Co-Authors: Emmanuelle Delagoutte, Elisabeth Bertrandburggraf, Iain B Lambert, Robert P P FuchsAbstract:Abstract Previous in vivo studies involving sequence 5′-CCCG 1 G 2 G 3 -3′ ( Sma I site) have demonstrated that adducts of N -2-acetylaminofluorene (AAF) to any of the three guanine residues of the Sma I sequence induce, with different efficiencies, two classes of −1 frameshift events, namely −G and −C mutations, referred to as targeted and semitargeted mutations, respectively. It has been proposed that both events occur during replication as a consequence of slippage events involving slipped mutagenic intermediates (SMIs). In order to evaluate the potential role of the UvrABC Excinuclease in frameshift mutagenesis, we have studied the interaction of this enzyme with DNA molecules mimicking SMIs in vitro . In all of our constructions, when present, the AAF adduct was located on the third guanine residue of the Sma I site (5′-CCCG 1 G 2 G 3 -3′). This strand was referred to as the top strand, the complementary strand being the bottom strand. Double-stranded heteroduplexes mimicking the targeted and semitargeted SMIs contained a deletion of a C and a G within the Sma I sequence in the bottom strand and were designated ΔC/3 and ΔG/3 when modified with the AAF on the third guanine residue in the top strand or ΔC/O and ΔG/O when unmodified. The modified homoduplex was designated Sma I/3. ΔC/O and ΔG/O were weakly recognized by UvrA 2 B, but not incised. All three AAF-modified substrates were recognized with similar efficiency and much more efficiently than unmodified heteroduplexes. With AAF-monomodified substrates, dissociation of UvrA 2 from the UvrA 2 B- DNA complex occurred more readily in heteroduplexes than in the homoduplex. Sma I/3 and ΔC/3 were incised with equal efficiency, while ΔG/3 was less incised. The position of the AAF lesion dictated the position of the incised phosphodiester bonds, suggesting that the presence of a bulge can modulate the yield but not the incision pattern of AAF-modified substrates. The finding that UvrABC Excinuclease acts on substrates that mimic SMIs suggests that the nucleotide excision repair pathway may help in fixing frameshift mutations before the following round of replication.
-
structure of uvrabc Excinuclease uv damaged dna complexes studied by flow linear dichroism dna curved by uvrb and uvrc
FEBS Letters, 1992Co-Authors: Masayuki Takahashi, Elisabeth Bertrandburggraf, Robert P P Fuchs, Bengt NordenAbstract:The interaction between UvrABC Excinuclease from Escherichia coli and ultraviolet light- (UV) damaged DNA was studied by flow linear dichroism. The dichroism signal from DNA was drastically decreased in intensity upon incubation with UvrA and UvrB or whole enzyme in the presence of effector ATP. The change was specific for UV-damaged DNA, and a concluded suppressed DNA orientation suggests the wrapping of DNA around the protein. The incubation with the UvrC subunit alone also somewhat reduces the signal, however, in this case the change was smaller and not specific for UV-damaged DNA. The structural modification of DNA, promoted by the (UvrA2-UvrB) complex, probably facilitates or stabilizes the interaction of the UvrC subunit with DNA for the excision.
