Evidence that the geneuvrB is indispensable for a polymerase I deficient strain ofEscherichia coli K-12

Morimyo, Mitsuoki; Shimazu, Yoshie

doi:10.1007/bf00582875

Cited by 25 publications

(14 citation statements)

References 25 publications

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“…More than 25 years ago it was proposed that UvrB and UvrD might also be involved in DNA replication, since in vivo in the absence of DNA damage-inducing treatments, uvrB or uvrD mutations were found to be lethal in combination with a mutation in the polA gene (11,29,38,39). Combining a deletion of the uvrB gene with either a polA1 or a polA12 mutation leads to inviability (38).…”

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“…Two different unidentified point mutations in uvrD are also lethal in combination with the polA1 mutation, indicating a role for UvrD in replication as well (11,39). In contrast with uvrB and uvrD, strains with point mutations in uvrA or uvrC (18,25,29,37) in a polA mutant background have been reported to be viable, although the plating efficiency of a uvrA6 polA12 strain was found to be reduced at 42°C (18,37).…”

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Role of the Escherichia coli Nucleotide Excision Repair Proteins in DNA Replication

2000

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DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication. Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, ⌬polA cells grow even better when the uvrC gene has been deleted. Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway. With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3 incision reaction. Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication. Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the uvrC gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB. A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro. The same mutation, however, does give inviability in combination with the ⌬polA mutation. Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair. A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed.In Escherichia coli, nucleotide excision repair (NER) is initiated by the action of the UvrA, UvrB, and UvrC proteins. The UvrA protein loads UvrB onto a damaged site, after which UvrC binds to UvrB, resulting in the UvrBC-DNA incision complex. In this complex, first an incision is made at the fourth or fifth phosphodiester bond on the 3Ј side of the damage, followed by incision at the eighth phosphodiester bond on the 5Ј side of the damage. Both incisions are catalyzed by the UvrC protein, which contains two distinct active sites, one for each incision (20,45). UvrD (helicase II) subsequently removes the damaged strand, and DNA polymerase I (PolI) fills in the resulting gap. Finally, the remaining nick is closed by DNA ligase (for reviews, see references 8 and 36).Besides its function in NER, it is generally believed that the major role of PolI in the cell is the processing of the lagging strand during DNA replication (16). In polA mutant strains the joining of Okazaki fragments is severely retarded (31,32,42). The protein possesses three enzymatic activities, a 5Ј-3Ј exonuclease activity located in the N-terminal part of the protein (the small domain) and a DNA polymerase activity which, together with a 3Ј-5Ј exonuclease activity, is located in the C-terminal part of the protein (the Klenow domain) (5, 15). The combination of the 5Ј-3Ј exonuclease and the polymerase activities results in the so-called nick translation ac...

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Role of the Escherichia coli Nucleotide Excision Repair Proteins in DNA Replication

2000

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“…Because both recB (13) and uvrB (14) mutations are also lethal when Poll is deficient, we constructed recB poUA12 and uvrB poUL12 double mutants and found that their unconditional inviabilities at 42°C were not alleviated by this plasmid. These negative results could mean either that overproduction of (6) and has been demonstrated with our strain, SC18-12 (16).…”

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Overproduction of DnaE protein (alpha subunit of DNA polymerase III) restores viability in a conditionally inviable Escherichia coli strain deficient in DNA polymerase I

Witkin

Roegner-Maniscalco

1992

J Bacteriol

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Table 1 compares the growth of the double-mutant strain SC18-12 with that of each of the single-mutant strains and with that of a poLA12 strain from which recA has been deleted. ThepoLA12 recA 718 strain differed from each of the single mutants at 42°C in its inability to form colonies on nutrient agar and in its extremely slow growth on minimal medium. Similar results were obtained at 37°C. The polA12 recA-deleted strain was unconditionally inviable at 42°C and grew very poorly at 30°C. Figure 1 shows that the rate of DNA synthesis on nutrient agar, which even at 30°C was slightly slower in the recA718poLA12 strain SC18-12 than in either single mutant, decreased steadily in the double mutant after 1 h at 42°C. The rates of DNA synthesis in both single mutants were indistinguishable from those in the recA+ poU+ strain SC18-RP under the same conditions (data not shown).

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“…It is likely that the 3'->5' exonuclease activity becomes pivotal when the SOS response is constitutively turned on, because polA 1 inactivates both polymerization and 3'-*5' exonuclease activities whereas poLA25::miniTnl0spc appears to impair polymerization activity only (4). recB and uvrB mutations are also lethal when combined with a defectivepoL4 gene (24,25). Whether these lethal combinations are also suppressed by the lexA(Def) mutation remains to be seen.…”

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DNA polymerase I and the bypassing of RecA dependence of constitutive stable DNA replication in Escherichia coli rnhA mutants

1993

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In Escherichia coli rnhA mutants, several normally repressed origins (oriK sites) of DNA replication are activated. The type of DNA replication initiated from these origins, termed constitutive stable DNA replication, does not require DnaA protein or the oriC site, which are essential for normal DNA replication. It requires active RecA protein. We previously found that the lexA71(Def)::Tn5 mutation can suppress this RecA requirement and postulated that the derepression of a LexA regulon gene(s) leads to the activation of a bypass pathway, Rip (for RecA-independent process). In this study, we isolated a miniTn10spc insertion mutant that abolishes the ability of the lexA(Def) mutation to suppress the RecA requirement of constitutive stable DNA replication. Cloning and DNA sequencing analysis of the mutant revealed that the insertion occurs at the 3' end of the coding region of the polA gene, which encodes DNA polymerase I. The mutant allele, designated polA25::miniTn10spc, is expected to abolish the polymerization activity but not the 5'-->3' or 3'-->5' exonuclease activity. Thus, the Rip bypass pathway requires active DNA polymerase I. Since the lethal combination of recA(Def) and polA25::miniTn10spc could be suppressed by derepression of the LexA regulon only when DNA replication is driven by the oriC system, it was suggested that the bypass pathway has a specific requirement for DNA polymerase I at the initiation step in the absence of RecA. An accompanying paper (Y. Cao and T. Kogoma, J. Bacteriol. 175:7254-7259, 1993) describes experiments to determine which activities of DNA polymerase I are required at the initiation step and discusses possible roles for DNA polymerase in the Rip bypass pathway.

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Evidence that the geneuvrB is indispensable for a polymerase I deficient strain ofEscherichia coli K-12

Cited by 25 publications

References 25 publications

Role of the Escherichia coli Nucleotide Excision Repair Proteins in DNA Replication

Role of the Escherichia coli Nucleotide Excision Repair Proteins in DNA Replication

Overproduction of DnaE protein (alpha subunit of DNA polymerase III) restores viability in a conditionally inviable Escherichia coli strain deficient in DNA polymerase I

DNA polymerase I and the bypassing of RecA dependence of constitutive stable DNA replication in Escherichia coli rnhA mutants

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