Targeting of the UmuD, UmuD', and MucA' mutagenesis proteins to DNA by RecA protein.

Frank, Ekaterina G.; Hauser, Janet; Levine, Arthur S.; Woodgate, Roger

doi:10.1073/pnas.90.17.8169

Cited by 100 publications

(102 citation statements)

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“…Umudependent error-prone translesion DNA synthesis is believed to occur as part of a multiprotein complex that requires protein interactions between UmuC and UmuD' (57), RecA (2,20,21,50), and probably subunits of DNA polymerase III holoenzyme (19,27,43,51). It is conceivable that the umuC mutants isolated here are defective in some, if not all, of the aforementioned interactions.…”

Section: Resultsmentioning

confidence: 99%

“…1). Plasmid pRW116 was constructed by replacing a 4-kb EcoRI fragment from pDS110, encoding kanamycin resistance and the structural umuDC operon, with a 2-kb EcoRI fragment carrying the genetically engineered umuD'C operon from plasmid pEC42 (20) (Fig. 1 Both the medium-copy-number plasmid pRW30 (55) and the low-copy-number plasmid pRW154 (24) that express the UmuDC proteins from their natural promoter have been described previously.…”

Section: Methodsmentioning

confidence: 99%

“…Mutants of umuD or recA that are unable to promote cleavage are rendered phenotypically nonmutable (2,18,31,46). RecA also participates directly in the mutagenic process by targeting the Umu-like mutagenesis proteins to DNA (13,20). Although recent studies have shown that DNA polymerase III holoenzyme can bypass a synthetic abasic lesion in vitro only in the presence of the highly purified UmuD'C and RecA proteins (43), the biochemical action of these proteins remains to be resolved.…”

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confidence: 99%

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Isolation and characterization of novel plasmid-encoded umuC mutants

Woodgate¹,

Singh²,

Kulaeva³

et al. 1994

J Bacteriol

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Interestingly, these plasmid mutants fell into two classes: (i) 5 were expression mutants that produced either too little or too much wild-type UmuC protein, and (ii) 11 were plasmids with structural changes in the UmuC protein. Although hydroxylamine mutagenesis was random, most of the structural mutants identified in the screen were localized to two regions of the UmuC protein; four mutations were found in a stretch of 30 amino acids (residues 133 to 162) in the middle of the protein, while four other mutations (three of which resulted in a truncated UmuC protein) were localized in the last 50 carboxyl-terminal amino acid residues. These new plasmid umuC mutants, together with the previously identified chromosomal umuC25, umuC36, and umuC104 mutations that we have also cloned, should prove extremel useful in dissecting the genetic and biochemical activities of UmuC in mutagenic DNA repair.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Isolation and characterization of novel plasmid-encoded umuC mutants

Woodgate¹,

Singh²,

Kulaeva³

et al. 1994

J Bacteriol

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“…This binding is mediated most likely through UmuDЈ, which was previously shown to interact with RecA (36). So far, there was no demonstration of a direct binding between UmuC and RecA.…”

Section: Requirement For Ssb In Lesion Bypass-ssb Was Previouslymentioning

confidence: 99%

Lesion Bypass by the Escherichia coli DNA Polymerase V Requires Assembly of a RecA Nucleoprotein Filament

Reuven

Arad

Stasiak

et al. 2001

Journal of Biological Chemistry

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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 SOSregulated proteins UmuD and RecA and the singlestrand 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. ATP␥S, 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.Genomic DNA is afflicted by numerous lesions that might interfere with its propagation and with gene expression (1). Most of these lesions, which are usually base modifications, are repaired by cellular DNA repair mechanisms (1). When the replication fork encounters a blocking DNA lesion that has escaped repair, replication stops forming a ssDNA 1 region in DNA (2). In Escherichia coli at least two mechanisms, which are regulated by the SOS response (3), act to repair the gap by converting the ssDNA region into a dsDNA region without actually removing the damaged nucleotide. Recombinational repair patches the gap with a complementary DNA segment from the fully replicated sister chromatid (4, 5), whereas translesion replication fills in the gap by DNA synthesis. This pathway, also termed lesion bypass or error-prone repair, is mutagenic, because DNA lesions often cause misincorporation by DNA polymerases, leading to the formation of mutations (2, 6).The in vitro reconstitution of SOS translesion replication with purified components (7-9) established that SOS-targeted mutagenesis occurs by replication through DNA lesions by DNA polymerase V (UmuC) 2 in the presence of UmuDЈ, RecA, and SSB (10, 11). Pol V effectively bypasses a synthetic abasic site (10, 11), a cyclobutyl TT dimer and a 6-4 TT adduct (12), leading to targeted mutations. When replicating an undamaged DNA template pol V is highly mutagenic and forms preferentially purine-purine and pyrimidine-pyrimidine mismatches, resulting in transversion mutations (13). These activities of pol V are responsible for SOS mutagenesis targeted to DNA lesions and for untargeted mutagenesis, which occurs in undamaged DNA regions.Proteins similar to UmuC are widespread from E. coli...

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“…First and foremost is the targeting mechanism, which for Pol V is mediated by the single-stranded DNAbinding protein SSB (P. Pham and M. F. Goodman, unpublished data) and RecA (Sweasy et al 1990;Bailone et al 1991;Frank et al 1993). Without RecA, Pol V has a very low probability of using a DNA template, a feature that limits Pol V activity only to areas of severe replication fork blockage (Tang et al 2000).…”

Section: Rad30b/pol I I I I I I I I I I I I As the Trans-acting Mutatmentioning

confidence: 99%

A new class of errant DNA polymerases provides candidates for somatic hypermutation

Tippin

Goodman

2001

Phil. Trans. R. Soc. Lond. B

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The mechanism of somatic hypermutation of the immunoglobulin genes remains a mystery after nearly 30 years of intensive research in the ¢eld. While many clues to the process have been discovered in terms of the genetic elements required in the immunoglobulin genes, the key enzymatic players that mediate the introduction of mutations into the variable region are unknown. The recent wave of newly discovered eukaryotic DNA polymerases have given a fresh supply of potential candidates and a renewed vigour in the search for the elusive mutator factor governing a¤nity maturation. In this paper, we discuss the relevant genetic and biochemical evidence known to date regarding both somatic hypermutation and the new DNA polymerases and address how the two ¢elds can be brought together to identify the strongest candidates for further study. In particular we discuss evidence for the in vitro biochemical misincorporation properties of human Rad30B/Pol i and how it compares to the in vivo somatic hypermutation spectra.

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Targeting of the UmuD, UmuD', and MucA' mutagenesis proteins to DNA by RecA protein.

Cited by 100 publications

References 37 publications

Isolation and characterization of novel plasmid-encoded umuC mutants

Isolation and characterization of novel plasmid-encoded umuC mutants

Lesion Bypass by the Escherichia coli DNA Polymerase V Requires Assembly of a RecA Nucleoprotein Filament

A new class of errant DNA polymerases provides candidates for somatic hypermutation

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