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Myron F. Goodman - One of the best experts on this subject based on the ideXlab platform.
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umud 2c is an error prone dna polymerase escherichia coli pol v
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Mengjia Tang, Ekaterina G Frank, Roger Woodgate, Mike Odonnell, Xuan Shen, Myron F. GoodmanAbstract:The damage-inducible UmuD′ and UmuC Proteins are required for most SOS mutagenesis in Escherichia coli. Our recent assay to reconstitute this process in vitro, using a native UmuD′2C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD′2C. E. coli dinB has recently been shown to have DNA polymerase activity (pol IV). We suggest that UmuD′2C, the fifth DNA polymerase discovered in E. coli, be designated as E. coli pol V. In the presence of RecA, β sliding clamp, γ clamp loading complex, and E. coli Single-Stranded Binding Protein (SSB), pol V’s polymerase activity is highly “error prone” at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE). Pol V bypasses a site-directed abasic lesion with an efficiency about 100- to 150-fold higher than pol III HE. In accordance with the “A-rule,” dAMP is preferentially incorporated opposite the lesion. A pol V mutant, UmuD′2C104 (D101N), has no measurable lesion bypass activity. A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3′-OH template-primer ends. We show, however, that despite competition for primer-3′-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion.
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Biochemical basis of SOS-induced mutagenesis in Escherichia coli: Reconstitution of in vitro lesion bypass dependent on the UmuD′2C mutagenic complex and RecA Protein
Proceedings of the National Academy of Sciences, 1998Co-Authors: Mengjia Tang, Mike O'donnell, Jennifer Turner, Irina Bruck, Ramon Eritja, Ekaterina G Frank, Roger Woodgate, Myron F. GoodmanAbstract:Damage-induced SOS mutagenesis requiring the UmuD′C Proteins occurs as part of the cells’ global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC Protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD′2C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD′2C, activated RecA Protein (RecA*), β-sliding clamp, γ-clamp loading complex, Single-Stranded Binding Protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD′2C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD′2C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.
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biochemical basis of sos induced mutagenesis in escherichia coli reconstitution of in vitro lesion bypass dependent on the umud 2c mutagenic complex and reca Protein
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Mengjia Tang, Jennifer Turner, Irina Bruck, Ramon Eritja, Ekaterina G Frank, Roger Woodgate, Mike Odonnell, Myron F. GoodmanAbstract:Damage-induced SOS mutagenesis requiring the UmuD′C Proteins occurs as part of the cells’ global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC Protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD′2C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD′2C, activated RecA Protein (RecA*), β-sliding clamp, γ-clamp loading complex, Single-Stranded Binding Protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD′2C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD′2C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.
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DYNAMICS OF LOADING THE BETA SLIDING CLAMP OF DNA POLYMERASE III ONTO DNA
Journal of Biological Chemistry, 1996Co-Authors: Linda B. Bloom, Mike O'donnell, Jennifer Turner, Zvi Kelman, Joseph M. Beechem, Myron F. GoodmanAbstract:Abstract A “minimal” DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the β sliding clamp, γ complex clamp loader, and Single-Stranded Binding Protein from Escherichia coli. This primer-template system was used to measure the loading of the β sliding clamp by the γ complex in an ATP-dependent reaction. Bound Protein-DNA complexes were detected by monitoring fluorescence depolarization of DNA. Steady state and time-resolved anisotropies were measured, and stopped-flow pre-steady state fluorescence measurements allowed visualization of the loading reactions in real time. The rate of loading β onto DNA was 12 s−1, demonstrating that clamp assembly is rapid on the time scale required for lagging strand Okazaki fragment synthesis. The association rate appears to be limited by an intramolecular step occurring prior to the clamp-loading reaction, possibly the opening of the toroidal β dimer.
Mengjia Tang - One of the best experts on this subject based on the ideXlab platform.
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umud 2c is an error prone dna polymerase escherichia coli pol v
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Mengjia Tang, Ekaterina G Frank, Roger Woodgate, Mike Odonnell, Xuan Shen, Myron F. GoodmanAbstract:The damage-inducible UmuD′ and UmuC Proteins are required for most SOS mutagenesis in Escherichia coli. Our recent assay to reconstitute this process in vitro, using a native UmuD′2C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD′2C. E. coli dinB has recently been shown to have DNA polymerase activity (pol IV). We suggest that UmuD′2C, the fifth DNA polymerase discovered in E. coli, be designated as E. coli pol V. In the presence of RecA, β sliding clamp, γ clamp loading complex, and E. coli Single-Stranded Binding Protein (SSB), pol V’s polymerase activity is highly “error prone” at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE). Pol V bypasses a site-directed abasic lesion with an efficiency about 100- to 150-fold higher than pol III HE. In accordance with the “A-rule,” dAMP is preferentially incorporated opposite the lesion. A pol V mutant, UmuD′2C104 (D101N), has no measurable lesion bypass activity. A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3′-OH template-primer ends. We show, however, that despite competition for primer-3′-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion.
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biochemical basis of sos induced mutagenesis in escherichia coli reconstitution of in vitro lesion bypass dependent on the umud 2c mutagenic complex and reca Protein
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Mengjia Tang, Jennifer Turner, Irina Bruck, Ramon Eritja, Ekaterina G Frank, Roger Woodgate, Mike Odonnell, Myron F. GoodmanAbstract:Damage-induced SOS mutagenesis requiring the UmuD′C Proteins occurs as part of the cells’ global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC Protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD′2C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD′2C, activated RecA Protein (RecA*), β-sliding clamp, γ-clamp loading complex, Single-Stranded Binding Protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD′2C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD′2C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.
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Biochemical basis of SOS-induced mutagenesis in Escherichia coli: Reconstitution of in vitro lesion bypass dependent on the UmuD′2C mutagenic complex and RecA Protein
Proceedings of the National Academy of Sciences, 1998Co-Authors: Mengjia Tang, Mike O'donnell, Jennifer Turner, Irina Bruck, Ramon Eritja, Ekaterina G Frank, Roger Woodgate, Myron F. GoodmanAbstract:Damage-induced SOS mutagenesis requiring the UmuD′C Proteins occurs as part of the cells’ global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC Protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD′2C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD′2C, activated RecA Protein (RecA*), β-sliding clamp, γ-clamp loading complex, Single-Stranded Binding Protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD′2C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD′2C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.
Roger Woodgate - One of the best experts on this subject based on the ideXlab platform.
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umud 2c is an error prone dna polymerase escherichia coli pol v
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Mengjia Tang, Ekaterina G Frank, Roger Woodgate, Mike Odonnell, Xuan Shen, Myron F. GoodmanAbstract:The damage-inducible UmuD′ and UmuC Proteins are required for most SOS mutagenesis in Escherichia coli. Our recent assay to reconstitute this process in vitro, using a native UmuD′2C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD′2C. E. coli dinB has recently been shown to have DNA polymerase activity (pol IV). We suggest that UmuD′2C, the fifth DNA polymerase discovered in E. coli, be designated as E. coli pol V. In the presence of RecA, β sliding clamp, γ clamp loading complex, and E. coli Single-Stranded Binding Protein (SSB), pol V’s polymerase activity is highly “error prone” at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE). Pol V bypasses a site-directed abasic lesion with an efficiency about 100- to 150-fold higher than pol III HE. In accordance with the “A-rule,” dAMP is preferentially incorporated opposite the lesion. A pol V mutant, UmuD′2C104 (D101N), has no measurable lesion bypass activity. A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3′-OH template-primer ends. We show, however, that despite competition for primer-3′-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion.
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biochemical basis of sos induced mutagenesis in escherichia coli reconstitution of in vitro lesion bypass dependent on the umud 2c mutagenic complex and reca Protein
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Mengjia Tang, Jennifer Turner, Irina Bruck, Ramon Eritja, Ekaterina G Frank, Roger Woodgate, Mike Odonnell, Myron F. GoodmanAbstract:Damage-induced SOS mutagenesis requiring the UmuD′C Proteins occurs as part of the cells’ global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC Protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD′2C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD′2C, activated RecA Protein (RecA*), β-sliding clamp, γ-clamp loading complex, Single-Stranded Binding Protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD′2C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD′2C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.
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Biochemical basis of SOS-induced mutagenesis in Escherichia coli: Reconstitution of in vitro lesion bypass dependent on the UmuD′2C mutagenic complex and RecA Protein
Proceedings of the National Academy of Sciences, 1998Co-Authors: Mengjia Tang, Mike O'donnell, Jennifer Turner, Irina Bruck, Ramon Eritja, Ekaterina G Frank, Roger Woodgate, Myron F. GoodmanAbstract:Damage-induced SOS mutagenesis requiring the UmuD′C Proteins occurs as part of the cells’ global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC Protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD′2C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD′2C, activated RecA Protein (RecA*), β-sliding clamp, γ-clamp loading complex, Single-Stranded Binding Protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD′2C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD′2C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.
Ekaterina G Frank - One of the best experts on this subject based on the ideXlab platform.
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umud 2c is an error prone dna polymerase escherichia coli pol v
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Mengjia Tang, Ekaterina G Frank, Roger Woodgate, Mike Odonnell, Xuan Shen, Myron F. GoodmanAbstract:The damage-inducible UmuD′ and UmuC Proteins are required for most SOS mutagenesis in Escherichia coli. Our recent assay to reconstitute this process in vitro, using a native UmuD′2C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD′2C. E. coli dinB has recently been shown to have DNA polymerase activity (pol IV). We suggest that UmuD′2C, the fifth DNA polymerase discovered in E. coli, be designated as E. coli pol V. In the presence of RecA, β sliding clamp, γ clamp loading complex, and E. coli Single-Stranded Binding Protein (SSB), pol V’s polymerase activity is highly “error prone” at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE). Pol V bypasses a site-directed abasic lesion with an efficiency about 100- to 150-fold higher than pol III HE. In accordance with the “A-rule,” dAMP is preferentially incorporated opposite the lesion. A pol V mutant, UmuD′2C104 (D101N), has no measurable lesion bypass activity. A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3′-OH template-primer ends. We show, however, that despite competition for primer-3′-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion.
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biochemical basis of sos induced mutagenesis in escherichia coli reconstitution of in vitro lesion bypass dependent on the umud 2c mutagenic complex and reca Protein
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Mengjia Tang, Jennifer Turner, Irina Bruck, Ramon Eritja, Ekaterina G Frank, Roger Woodgate, Mike Odonnell, Myron F. GoodmanAbstract:Damage-induced SOS mutagenesis requiring the UmuD′C Proteins occurs as part of the cells’ global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC Protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD′2C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD′2C, activated RecA Protein (RecA*), β-sliding clamp, γ-clamp loading complex, Single-Stranded Binding Protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD′2C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD′2C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.
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Biochemical basis of SOS-induced mutagenesis in Escherichia coli: Reconstitution of in vitro lesion bypass dependent on the UmuD′2C mutagenic complex and RecA Protein
Proceedings of the National Academy of Sciences, 1998Co-Authors: Mengjia Tang, Mike O'donnell, Jennifer Turner, Irina Bruck, Ramon Eritja, Ekaterina G Frank, Roger Woodgate, Myron F. GoodmanAbstract:Damage-induced SOS mutagenesis requiring the UmuD′C Proteins occurs as part of the cells’ global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC Protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD′2C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD′2C, activated RecA Protein (RecA*), β-sliding clamp, γ-clamp loading complex, Single-Stranded Binding Protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD′2C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD′2C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.
Mike Odonnell - One of the best experts on this subject based on the ideXlab platform.
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umud 2c is an error prone dna polymerase escherichia coli pol v
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Mengjia Tang, Ekaterina G Frank, Roger Woodgate, Mike Odonnell, Xuan Shen, Myron F. GoodmanAbstract:The damage-inducible UmuD′ and UmuC Proteins are required for most SOS mutagenesis in Escherichia coli. Our recent assay to reconstitute this process in vitro, using a native UmuD′2C complex, revealed that the highly purified preparation contained DNA polymerase activity. Here we eliminate the possibility that this activity is caused by a contaminating DNA polymerase and show that it is intrinsic to UmuD′2C. E. coli dinB has recently been shown to have DNA polymerase activity (pol IV). We suggest that UmuD′2C, the fifth DNA polymerase discovered in E. coli, be designated as E. coli pol V. In the presence of RecA, β sliding clamp, γ clamp loading complex, and E. coli Single-Stranded Binding Protein (SSB), pol V’s polymerase activity is highly “error prone” at both damaged and undamaged DNA template sites, catalyzing efficient bypass of abasic lesions that would otherwise severely inhibit replication by pol III holoenzyme complex (HE). Pol V bypasses a site-directed abasic lesion with an efficiency about 100- to 150-fold higher than pol III HE. In accordance with the “A-rule,” dAMP is preferentially incorporated opposite the lesion. A pol V mutant, UmuD′2C104 (D101N), has no measurable lesion bypass activity. A kinetic analysis shows that addition of increasing amounts of pol III to a fixed level of pol V inhibits lesion bypass, demonstrating that both enzymes compete for free 3′-OH template-primer ends. We show, however, that despite competition for primer-3′-ends, pol V and pol III HE can nevertheless interact synergistically to stimulate synthesis downstream from a template lesion.
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biochemical basis of sos induced mutagenesis in escherichia coli reconstitution of in vitro lesion bypass dependent on the umud 2c mutagenic complex and reca Protein
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Mengjia Tang, Jennifer Turner, Irina Bruck, Ramon Eritja, Ekaterina G Frank, Roger Woodgate, Mike Odonnell, Myron F. GoodmanAbstract:Damage-induced SOS mutagenesis requiring the UmuD′C Proteins occurs as part of the cells’ global response to DNA damage. In vitro studies on the biochemical basis of SOS mutagenesis have been hampered by difficulties in obtaining biologically active UmuC Protein, which, when overproduced, is insoluble in aqueous solution. We have circumvented this problem by purifying the UmuD′2C complex in soluble form and have used it to reconstitute an SOS lesion bypass system in vitro. Stimulated bypass of a site-directed model abasic lesion occurs in the presence of UmuD′2C, activated RecA Protein (RecA*), β-sliding clamp, γ-clamp loading complex, Single-Stranded Binding Protein (SSB), and either DNA polymerases III or II. Synthesis in the presence of UmuD′2C is nonprocessive on damaged and undamaged DNA. No lesion bypass is observed when wild-type RecA is replaced with RecA1730, a mutant that is specifically defective for Umu-dependent mutagenesis. Perhaps the most noteworthy property of UmuD′2C resides in its ability to stimulate both nucleotide misincorporation and mismatch extension at aberrant and normal template sites. These observations provide a biochemical basis for the role of the Umu complex in SOS-targeted and SOS-untargeted mutagenesis.