Lesion bypass by the Escherichia coli DNA polymerase V requires assembly of a RecA nucleoprotein filament.

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.

Highly mutagenic replication by DNA polymerase V (UmuC) provides a mechanistic basis for SOS untargeted mutagenesis.

When challenged by DNA-damaging agents, Escherichia coli cells respond by inducing the SOS stress response, which leads to an increase in mutation frequency by two mechanisms: translesion replication, a process that causes mutations because of misinsertion opposite the lesions, and an inducible mutator activity, which acts at undamaged sites. Here we report that DNA polymerase V (pol V; UmuC), which previously has been shown to be a lesion-bypass DNA polymerase, was highly mutagenic during in vitro gap-filling replication of a gapped plasmid carrying the cro reporter gene. This reaction required, in addition to pol V, UmuD', RecA, and single-stranded DNA (ssDNA)-binding protein. pol V produced point mutations at a frequency of 2.1 x 10(-4) per nucleotide (2.1% per cro gene), 41-fold higher than DNA polymerase III holoenzyme. The mutational spectrum of pol V was dominated by transversions (53%), which were formed at a frequency of 1.3 x 10(-4) per nucleotide (1. 1% per cro gene), 74-fold higher than with pol III holoenzyme. The prevalence of transversions and the protein requirements of this system are similar to those of in vivo untargeted mutagenesis (SOS mutator activity). This finding suggests that replication by pol V, in the presence of UmuD', RecA, and ssDNA-binding protein, is the basis of chromosomal SOS untargeted mutagenesis.

The mutagenesis protein UmuC is a DNA polymerase activated by UmuD', RecA, and SSB and is specialized for translesion replication.

Replication of DNA lesions leads to the formation of mutations. In Escherichia coli this process is regulated by the SOS stress response, and requires the mutagenesis proteins UmuC and UmuD'. Analysis of translesion replication using a recently reconstituted in vitro system (Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199) revealed that lesion bypass occurred with a UmuC fusion protein, UmuD', RecA, and SSB in the absence of added DNA polymerase. Further analysis revealed that UmuC was a DNA polymerase (E. coli DNA polymerase V), with a weak polymerizing activity. Upon addition of UmuD', RecA, and SSB, the UmuC DNA polymerase was greatly activated, and replicated a synthetic abasic site with great efficiency (45% bypass in 6 min), 10-100-fold higher than E. coli DNA polymerases I, II, or III holoenzyme. Analysis of bypass products revealed insertion of primarily dAMP (69%), and to a lesser degree dGMP (31%) opposite the abasic site. The UmuC104 mutant protein was defective both in lesion bypass and in DNA synthesis. These results indicate that UmuC is a UmuD'-, RecA-, and SSB-activated DNA polymerase, which is specialized for lesion bypass. UmuC is a member of a new family of DNA polymerases which are specialized for lesion bypass, and include the yeast RAD30 and the human XP-V genes, encoding DNA polymerase eta.

The beta subunit sliding DNA clamp is responsible for unassisted mutagenic translesion replication by DNA polymerase III holoenzyme.

The replication of damaged nucleotides that have escaped DNA repair leads to the formation of mutations caused by misincorporation opposite the lesion. In Escherichia coli, this process is under tight regulation of the SOS stress response and is carried out by DNA polymerase III in a process that involves also the RecA, UmuD' and UmuC proteins. We have shown that DNA polymerase III holoenzyme is able to replicate, unassisted, through a synthetic abasic site in a gapped duplex plasmid. Here, we show that DNA polymerase III*, a subassembly of DNA polymerase III holoenzyme lacking the beta subunit, is blocked very effectively by the synthetic abasic site in the same DNA substrate. Addition of the beta subunit caused a dramatic increase of at least 28-fold in the ability of the polymerase to perform translesion replication, reaching 52% bypass in 5 min. When the ssDNA region in the gapped plasmid was extended from 22 nucleotides to 350 nucleotides, translesion replication still depended on the beta subunit, but it was reduced by 80%. DNA sequence analysis of translesion replication products revealed mostly -1 frameshifts. This mutation type is changed to base substitution by the addition of UmuD', UmuC, and RecA, as demonstrated in a reconstituted SOS translesion replication reaction. These results indicate that the beta subunit sliding DNA clamp is the major determinant in the ability of DNA polymerase III holoenzyme to perform unassisted translesion replication and that this unassisted bypass produces primarily frameshifts.

The mutagenesis proteins UmuD' and UmuC prevent lethal frameshifts while increasing base substitution mutations.

Error-prone DNA repair consists of replicative filling-in of DNA gaps carrying lesions. We have reconstituted E. coli SOS error-prone repair using purified DNA polymerase III holoenzyme, SSB, RecA, UmuD', a UmuC fusion protein, and a gap lesion plasmid. In the absence of UmuDC, or without SOS induction, replication skips over the lesion, forming mostly one-nucleotide deletions. These cause translational frameshifts that usually inactivate genes. UmuD' and UmuC, in the presence of RecA and SSB, stimulate translesion replication and change its mutagenic specificity such that deletions are prevented and base substitutions are increased. This results in mutagenic but nondetrimental gap repair and provides an effective mechanism for generating genetic variation in bacteria adapting to environmental stress.

Functional overlap of tRNA nucleotidyltransferase, poly(A) polymerase I, and polynucleotide phosphorylase.

Repair of the 3'-terminal -CCA sequence of tRNA generally requires the action of the enzyme tRNA nucleotidyltransferase. However, in Escherichia coli in the absence of this enzyme, a decreased level of tRNA end repair continues. To ascertain the enzymes responsible for this residual repair, mutant strains were constructed lacking tRNA nucleotidyltransferase and other enzymes potentially involved in the process, poly(A) polymerase I and polynucleotide phosphorylase (PNPase). Strains lacking tRNA nucleotidyltransferase and either one of the other enzymes displayed decreased growth rates and increased levels of defective tRNA compared with the single cca mutant. Triple mutants lacking all three enzymes grew very slowly, had even more defective tRNA, and were devoid of activity incorporating AMP into tRNA-C-C. Overexpression of poly(A) polymerase I, but not PNPase, partially compensated for the absence of tRNA nucleotidyltransferase. These data show that poly(A) polymerase I and PNPase participate in the end repair process and are required to maintain functional tRNA levels when tRNA nucleotidyltransferase is absent.

The gene for the longest known Escherichia coli protein is a member of helicase superfamily II.

The Escherichia coli rnt gene, which encodes the RNA-processing enzyme RNase T, is cotranscribed with a downstream gene. Complete sequencing of this gene indicates that its coding region encompasses 1,538 amino acids, making it the longest known protein in E. coli. The gene (tentatively termed lhr for long helicase related) contains the seven conserved motifs of the DNA and RNA helicase superfamily II. An approximately 170-kDa protein is observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 35S-labeled extracts prepared from cells in which lhr is under the control of an induced T7 promoter. This protein is absent when lhr is interrupted or when no plasmid is present. Downstream of lhr is the C-terminal region of a convergent gene with homology to glutaredoxin. Interruptions of chromosomal lhr at two different positions within the gene do not affect the growth of E. coli at various temperatures in rich or minimal medium, indicating that lhr is not essential for usual laboratory growth. lhr interruption also has no effect on anaerobic growth. In addition, cells lacking Lhr recover normally from starvation, plate phage normally, and display normal sensitivities to UV irradiation and H2O2. Southern analysis showed that no other gene closely related to lhr is present on the E. coli chromosome. These data expand the known size range of E. coli proteins and suggest that very large helicases are present in this organism.

Substitution of the 3' terminal adenosine residue of transfer RNA in vivo.

We have altered by site-directed mutagenesis the 3' terminal adenosine residue of a tRNA(Tyrsu3+) gene encoded on a single-copy plasmid and examined the consequences of these substitutions on suppressor activity in vivo. Our data show that mutant su3 genes containing 3'-CCC, -CCG, or -CCU termini instead of -CCA can be efficiently transcribed and processed in Escherichia coli to generate functional suppressor tRNAs. However, in contrast to normal tRNA genes, both tRNA nucleotidyltransferase and exoribonuclease activities are required to obtain suppression by the mutant tRNAs, indicating that removal of the incorrect 3' terminal residue and resynthesis of the normal -CCA terminus are occurring in this situation. In addition, a low level of suppressor activity and tRNA repair was found in cells devoid of tRNA nucleotidyltransferase, suggesting that an additional activity able to partially repair the 3' end of tRNA is present in E. coli. The use of mutant strains lacking one or several exoribonucleases revealed that the various RNAses have very different specificities for removal of incorrect 3' residues and that these differ greatly from their action on CCA-ending tRNA. These data show that the 3' terminal adenosine residue is necessary for tRNA function in vivo and that cells can compensate for its alteration by changes in the normal pathway of tRNA metabolism.

Multiple exoribonucleases are required for the 3' processing of Escherichia coli tRNA precursors in vivo.

Our knowledge of the 3' processing of tRNA precursors is severely limited. Although six exoribonucleases able to act on Escherichia coli tRNA precursors in vitro have been identified, their involvement in tRNA maturation in vivo has not been demonstrated. Here we show, using a wide range of multiple RNase-deficient strains and a quantitative suppression assay, that at least five of these enzymes--RNase II, RNase D, RNase BN, RNase T, and RNase PH--can participate in the synthesis of functional tRNA(Tyr)su+3 in vivo. Moreover, any one of the five RNases is sufficient to allow tRNA processing to proceed although with varying effectiveness. Examination of the level of aminoacylation of tRNA isolated from RNase-deficient strains suggested that tRNA precursors accumulate in the most defective cells. These data indicate that exoribonucleases are required for tRNA maturation in vivo and that there is a high degree of functional overlap among the enzymes. These studies contribute to the identification of all the enzymes necessary for defining the complete processing pathway for E. coli tRNA precursors.

RNase PH is essential for tRNA processing and viability in RNase-deficient Escherichia coli cells.

RNase PH is a Pi-dependent exoribonuclease that can act at the 3' terminus of tRNA precursors in vitro. To obtain information about the function of this enzyme in vivo, the Escherichia coli rph gene encoding RNase PH was interrupted with either a kanamycin resistance or a chloramphenicol resistance cassette and transferred to the chromosome of a variety of RNase-resistant strains. Inactivation of the chromosomal copy of rph eliminated RNase PH activity from extracts and also slowed the growth of many of the strains, particularly ones that already were deficient in RNase T or polynucleotide phosphorylase. Introduction of the rph mutation into a strain already lacking RNases I, II, D, BN, and T resulted in inviability. The rph mutation also had dramatic effects on tRNA metabolism. Using an in vivo suppressor assay we found that elimination of RNase PH greatly decreased the level of su3+ activity in cells deficient in certain of the other RNases. Moreover, in an in vitro tRNA processing system the defect caused by elimination of RNase PH was shown to be the accumulation of a precursor that contained 4-6 additional 3' nucleotides following the -CCA sequence. These data indicate that RNase PH can be an essential enzyme for the processing of tRNA precursors.

Enzymatic basis for hydrolytic versus phosphorolytic mRNA degradation in Escherichia coli and Bacillus subtilis.

The rapid synthesis and breakdown of mRNA in prokaryotes can impose a significant energy drain on these cells. Previous in vivo studies [Duffy, J. J., Chaney, S. G. & Boyer, P. D. (1972) J. Mol. Biol. 64, 565-579; Chaney, S. G. & Boyer, P. D. (1972) J. Mol. Biol. 64, 581-591] indicated that while RNA turnover in Escherichia coli was hydrolytic, it was nonhydrolytic in Bacillus subtilis. Here we provide an explanation for these observations based on enzymatic analysis of extracts of these two organisms. RNA degradation to the mononucleotide level in E. coli extracts is due solely to two active ribonucleases, RNase II and polynucleotide phosphorylase, which act hydrolytically and phosphorolytically, respectively. RNase II activity represents close to 90% of the total activity of the extract, as expected for predominantly hydrolytic degradation in this organism. In contrast, RNase II is absent from B. subtilis extracts, and the primary mode of RNA degradation is phosphorolytic, employing the Bacillus equivalent of polynucleotide phosphorylase and releases nucleoside diphosphates as products. A low level of a Mn2(+)-stimulated, hydrolytic ribonuclease is also detectable in B. subtilis extracts. Overall, E. coli and B. subtilis extracts differ by about 20- to 100-fold, depending on the substrate, in their relative use of hydrolytic and phosphorolytic routes of RNA degradation. The relation of the mode of mRNA degradation to the environment of the cell is discussed.