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

Abstract

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 lesionbypass 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 ؋ 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 ؋ 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 SOS stress response is induced in Escherichia coli by single-stranded DNA (ssDNA) gaps formed when DNA lesions that have escaped repair block replication (1-3). Unable to remove the lesion from such gap͞lesion structures, the cells activate a tolerance response, which involves filling in the DNA gaps without removal of the lesion, thereby restoring genome continuity. Thereafter, a second attempt to eliminate the lesion by DNA repair can be made. Filling in of the gap is done by patching of a homologous DNA segment from the fully replicated sister chromatid via recombinational repair (4, 5) or by translesion replication, which requires the SOS-inducible proteins UmuC, UmuDЈ, and RecA (6, 7). The latter process is mutagenic, because of the miscoding promoted by most DNA lesions. Recently, it was found that UmuC is a DNA polymerase, termed DNA polymerase V (pol V), with a remarkable capability to replicate through DNA lesions that severely block other DNA polymerases (8,9).In addition to this mutagenesis process, which is targeted to DNA lesions, a mutator activity is induced under SOS conditions, which produces mutations in the apparent absence of DNA damage (untargeted mutagenesis) (10-12). Chromosomal untargeted mutagenesis requires the SOS-inducible proteins RecA, UmuDЈ, and UmuC (1, 13, 14), the same proteins that are required for translesion replication. In addition, it exhibits a particular mutational specificity, namely, the selective generation of transversions (14-17). Another pathway of untargeted mutagenesis is observed by transfecting UV-irradiated E. coli cells with unirradiated phage (18). This phage untargeted mutagenesis requires the dinB, uvrA, and polA gene products ...

Posttranslational modification of the umuD -encoded subunit of Escherichia coli DNA polymerase V regulates its interactions with the β processivity clamp

“…UmuDЈ does not act in the DNA damage checkpoint control, but rather participates in the second role of the umuDC gene products. The UmuDЈ 2 homodimer interacts with UmuC, forming the UmuDЈ 2 C complex (pol V), that functions as a lesion-bypass DNA polymerase to enable translesion DNA synthesis (TLS) (11,12,23,24). Viewed in this way, posttranslational modification of UmuD to yield UmuDЈ acts as a molecular switch to regulate temporally the two physiological roles of the umuDC gene products.…”

SOS mutator activity: Unequal mutagenesis on leading and lagging strands

“…This stalling provides an entry point for the constitutively present SOS proteins, leading to increased probability of mismatch extension and, hence, mutation. In view of the recent discovery of a polymerase activity for UmuC or UmuD 2 C (pol V) (3)(4)(5)(6), it is likely that this polymerase is responsible for the mismatch extension. However, the exact chain of events leading to mismatch extension, including the precise exchange and interplay between pol III and pol V, remains to be elucidated.…”

“…The UmuDЈ 2 C complex then acts jointly with RecA protein, which also has a direct role in the process, to create mutations at the sites of DNA lesions (targeted mutagenesis). The UmuDЈ 2 C complex has recently been demonstrated to possess a polymerase activity (pol V) (3)(4)(5)(6), which likely plays a critical role in producing the mutation. UmuC is an example of a specialized polymerase, a number of which have been reported from a variety of organisms and which are generally thought to function in mutagenesis and DNA damage processing (for reviews, see refs.…”

The many faces of DNA polymerases: Strategies for mutagenesis and for mutational avoidance

“…This protein complexes with UmuDЈ protein (a proteolytically processed form of UmuD) to form a UmuDЈ 2 C complex that facilitates TLS in the presence of E. coli DNA polymerase III holoenzyme (1)(2)(3)(4)(5)13). The UmuDЈ 2 C complex alone (most likely the UmuC polypeptide) is now called DNA polymerase V of E. coli (16)(17)(18). More recent studies have shown that DNA polymerase V bypasses thymine-thymine dimers, photoproducts, and abasic sites in a highly error-prone manner in vitro (19).…”