In vitro inhibition of RecA-mediated homologous pairing by UmuD'C proteins

Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions

“…Our observation that cells harboring the o C 1 version of UmuD 8 S60A are extremely sensitive to UV light suggests that elevated levels of UmuD 8 S60A can be harmful to cells. We next examined another characteristic phenotype of umuDC , specifically the inhibition of RecA-mediated homologous recombination by elevated levels of UmuDʹC [42–47]. UmuD 8 and UmuD 18 show similar levels of inhibition of RecA-mediated homologous recombination as full-length UmuD, again indicating that the truncated proteins appear to be proficient for interaction with RecA.…”

Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions

“…Our observation that cells harboring the o C 1 version of UmuD 8 S60A are extremely sensitive to UV light suggests that elevated levels of UmuD 8 S60A can be harmful to cells. We next examined another characteristic phenotype of umuDC , specifically the inhibition of RecA-mediated homologous recombination by elevated levels of UmuDʹC [42–47]. UmuD 8 and UmuD 18 show similar levels of inhibition of RecA-mediated homologous recombination as full-length UmuD, again indicating that the truncated proteins appear to be proficient for interaction with RecA.…”

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

“…Variants in the N-terminal region of loop 1 partially suppress inhibition of RecA-mediated homologous recombination. UmuDЈ and UmuC inhibit RecA-mediated homologous recombination when present at elevated levels, such as in the SOS response, which may be important for regulating RecA and its involvement in cellular processes (10,56,68,70,74,76). UmuC variants N32A, N33A, and D34A were expressed from the respective derivatives of plasmid pGY9738 in a ⌬umuDC strain.…”

Genetic and Biochemical Characterization of a Novel umuD Mutation: Insights into a Mechanism for UmuD Self-Cleavage

“…Although other recA alleles shown to be similarly refractory to the inhibitory effect of elevated levels of UmuDЈ 2 C were less active in SOS mutagenesis (60), it is possible that their defect in SOS mutagenesis was due to a deficiency of the mutant RecA proteins in TLS rather than to their reduced sensitivity to UmuDЈ 2 C. Recent electron microscopy studies have suggested that UmuDЈ 2 C binds preferentially to the tip of the RecA-ssDNA filament but at higher levels can also bind within the helical groove of the filament (13). Based on this and other findings (53,59,60,63), it has been suggested that the binding of UmuDЈ 2 C to the helical groove might competitively inhibit RecA-mediated homologous recombination, while its interaction with the tip of the RecA-ssDNA filament might deliver UmuDЈ 2 C to the site of the lesion. Further work will be required to see whether the UmuDЈ1012 protein is affected in its interaction with the groove and/or the tip of RecA-ssDNA nucleoprotein filaments.…”

“…In addition to its role in TLS, elevated levels of UmuDЈ together with UmuC act to antagonize RecA-mediated homologous recombination in vivo (59). It has been suggested that this inhibition of homologous recombina- tion might result from an effect on the formation of RecA-ssDNA filaments and may constitute a general mechanism by which RecA-mediated homologous recombination is attenuated to allow TLS (59,63). Our finding that umuDЈ1012 is able to efficiently promote SOS mutagenesis, despite its inability to efficiently inhibit RecA-mediated homologous recombination in vivo, indicates that this inhibition of recombination is not a prerequisite for SOS mutagenesis under our experimental conditions.…”

Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions

“…Our observation that cells harboring the o C 1 version of UmuD 8 S60A are extremely sensitive to UV light suggests that elevated levels of UmuD 8 S60A can be harmful to cells. We next examined another characteristic phenotype of umuDC , specifically the inhibition of RecA-mediated homologous recombination by elevated levels of UmuDʹC [42–47]. UmuD 8 and UmuD 18 show similar levels of inhibition of RecA-mediated homologous recombination as full-length UmuD, again indicating that the truncated proteins appear to be proficient for interaction with RecA.…”

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

“…Variants in the N-terminal region of loop 1 partially suppress inhibition of RecA-mediated homologous recombination. UmuDЈ and UmuC inhibit RecA-mediated homologous recombination when present at elevated levels, such as in the SOS response, which may be important for regulating RecA and its involvement in cellular processes (10,56,68,70,74,76). UmuC variants N32A, N33A, and D34A were expressed from the respective derivatives of plasmid pGY9738 in a ⌬umuDC strain.…”

Genetic and Biochemical Characterization of a Novel umuD Mutation: Insights into a Mechanism for UmuD Self-Cleavage

“…Although other recA alleles shown to be similarly refractory to the inhibitory effect of elevated levels of UmuDЈ 2 C were less active in SOS mutagenesis (60), it is possible that their defect in SOS mutagenesis was due to a deficiency of the mutant RecA proteins in TLS rather than to their reduced sensitivity to UmuDЈ 2 C. Recent electron microscopy studies have suggested that UmuDЈ 2 C binds preferentially to the tip of the RecA-ssDNA filament but at higher levels can also bind within the helical groove of the filament (13). Based on this and other findings (53,59,60,63), it has been suggested that the binding of UmuDЈ 2 C to the helical groove might competitively inhibit RecA-mediated homologous recombination, while its interaction with the tip of the RecA-ssDNA filament might deliver UmuDЈ 2 C to the site of the lesion. Further work will be required to see whether the UmuDЈ1012 protein is affected in its interaction with the groove and/or the tip of RecA-ssDNA nucleoprotein filaments.…”

“…In addition to its role in TLS, elevated levels of UmuDЈ together with UmuC act to antagonize RecA-mediated homologous recombination in vivo (59). It has been suggested that this inhibition of homologous recombina- tion might result from an effect on the formation of RecA-ssDNA filaments and may constitute a general mechanism by which RecA-mediated homologous recombination is attenuated to allow TLS (59,63). Our finding that umuDЈ1012 is able to efficiently promote SOS mutagenesis, despite its inability to efficiently inhibit RecA-mediated homologous recombination in vivo, indicates that this inhibition of recombination is not a prerequisite for SOS mutagenesis under our experimental conditions.…”

Altering the N-terminal arms of the polymerase manager protein UmuD modulates protein interactions

“…Our observation that cells harboring the o C 1 version of UmuD 8 S60A are extremely sensitive to UV light suggests that elevated levels of UmuD 8 S60A can be harmful to cells. We next examined another characteristic phenotype of umuDC , specifically the inhibition of RecA-mediated homologous recombination by elevated levels of UmuDʹC [42–47]. UmuD 8 and UmuD 18 show similar levels of inhibition of RecA-mediated homologous recombination as full-length UmuD, again indicating that the truncated proteins appear to be proficient for interaction with RecA.…”

Characterization of Escherichia coli UmuC Active-Site Loops Identifies Variants That Confer UV Hypersensitivity

“…Variants in the N-terminal region of loop 1 partially suppress inhibition of RecA-mediated homologous recombination. UmuDЈ and UmuC inhibit RecA-mediated homologous recombination when present at elevated levels, such as in the SOS response, which may be important for regulating RecA and its involvement in cellular processes (10,56,68,70,74,76). UmuC variants N32A, N33A, and D34A were expressed from the respective derivatives of plasmid pGY9738 in a ⌬umuDC strain.…”

Genetic and Biochemical Characterization of a Novel umuD Mutation: Insights into a Mechanism for UmuD Self-Cleavage

“…Although other recA alleles shown to be similarly refractory to the inhibitory effect of elevated levels of UmuDЈ 2 C were less active in SOS mutagenesis (60), it is possible that their defect in SOS mutagenesis was due to a deficiency of the mutant RecA proteins in TLS rather than to their reduced sensitivity to UmuDЈ 2 C. Recent electron microscopy studies have suggested that UmuDЈ 2 C binds preferentially to the tip of the RecA-ssDNA filament but at higher levels can also bind within the helical groove of the filament (13). Based on this and other findings (53,59,60,63), it has been suggested that the binding of UmuDЈ 2 C to the helical groove might competitively inhibit RecA-mediated homologous recombination, while its interaction with the tip of the RecA-ssDNA filament might deliver UmuDЈ 2 C to the site of the lesion. Further work will be required to see whether the UmuDЈ1012 protein is affected in its interaction with the groove and/or the tip of RecA-ssDNA nucleoprotein filaments.…”

“…In addition to its role in TLS, elevated levels of UmuDЈ together with UmuC act to antagonize RecA-mediated homologous recombination in vivo (59). It has been suggested that this inhibition of homologous recombina- tion might result from an effect on the formation of RecA-ssDNA filaments and may constitute a general mechanism by which RecA-mediated homologous recombination is attenuated to allow TLS (59,63). Our finding that umuDЈ1012 is able to efficiently promote SOS mutagenesis, despite its inability to efficiently inhibit RecA-mediated homologous recombination in vivo, indicates that this inhibition of recombination is not a prerequisite for SOS mutagenesis under our experimental conditions.…”