Bypass and termination at apurinic sites during replication of single-stranded DNA in vitro: a model for apurinic site mutagenesis.

Abstract

Mutations produced in Escherichia coli by apurinic sites are believed to arise via SOS-assisted translesion replication. Analysis of replication products synthesized on depurinated single-stranded DNA by DNA polymerase Ill holoenzyme revealed that apurinic sites frequently blocked in vitro replication. Bypass frequency of an apurinic site was estimated to be 10-15%. Direct evidence for replicative bypass was obtained in a complete single-stranded -+ replicative form replication system containing DNA polymerase III holoenzyme, single-stranded DNA binding protein, DNA polymerase I, and DNA ligase, by demonstrating the sensitivity of fully replicated products to the apurinic endonuclease activity of E. coli exonuclease III. Termination at apurinic sites, like termination at pyrimidine photodimers, involved dissociation of the polymerase from the blocked termini, followed by initiations at available primer templates. When no regular primer templates were available, the polymerase underwent repeated cycles of dissociation and rebinding at the blocked termini and, while bound, carried out multiple polymerization-excision reactions opposite the apurinic sites, leading to turnover of dNTPs into dNMPs. From the in vitro turnover rates, we could predict with striking accuracy the specificity of apurinic site mutagenesis, as determined in vivo in depurinated single-stranded DNA from an M13-ac hybrid phage. This fimding is consistent with the view that DNA polymerase III holoenzyme carries out the mutagenic "misinsertion" step during apurinic site mutagenesis in vivo and that the specificity of the process is determined primarily by the polymerase. SOS-induced proteins such as UmuD/C might act as processivity-like factors to stabilize the polymerase-DNA complex, thus increasing the efficiency of the next stage of past-lesion polymerization required to complete the bypass reaction.Apurinic (AP) and apyrimidinic sites are common lesions in DNA and are believed to be important intermediates in chemical carcinogenesis by a variety of chemical agents (1,2). Depurination is the most frequent spontaneous alteration in DNA under physiological conditions and occurs in vitro at the rate of 3 x 10-11 per nucleotide per sec (3). Extrapolation to the in vivo situation would suggest that 0.5 purine is lost from an Escherichia coli cell per generation, and as many as 10,000 purines are lost in each mammalian cell per day (3). Depyrimidination is much slower and occurs at rates 100 times lower than depurination (4). Exposure of cells to a variety of carcinogens leads to the formation of modified bases, some of which are converted to AP sites either because of enhanced spontaneous release (e.g., 7-methylguanine) (5) or by specific DNA glycosylases (e.g., 3-methyladenine) as part of the DNA repair processes (6). Specific DNA glycosylases form AP sites also by removing uracils or hypoxanthines misincorporated during replication or produced via deamination of cytosines or adenines, respectively (6).In E. coli, AP sites are highly mutag...

The Mutagenesis Protein MucB Interacts with Single Strand DNA Binding Protein and Induces a Major Conformational Change in Its Complex with Single-stranded DNA

“…3), whereas in vitro bypass of blocking lesions (2,(11)(12)(13)(14)(15)(16)(17)(18)(19) and in vitro UV mutagenesis (20 -22) can occur without these proteins. The report on umuC-independent UV mutagenesis in phage S13 (55) and the finding that umuC-independent UV mutagenesis is observed when a screening rather than selection procedure is used (56) support the view that UmuDЈC stimulates the mutagenic reaction, rather than being absolutely required.…”

“…A prevailing hypothesis is that UmuDЈ, the active form of UmuD (7)(8)(9), along with UmuC are required to assist the DNA polymerase in replicating the damaged site (10). However, the nature of this assistance is not clear because purified DNA polymerases can bypass DNA lesions unassisted (11)(12)(13)(14)(15)(16)(17)(18)(19). Moreover, in an in vitro system for UV mutagenesis carried out with crude protein extracts (20,21) or with purified proteins (22), we have found that UV mutations were effectively produced in the absence of UmuDЈ and UmuC.…”

Levels of epsilon, an essential replication subunit of Escherichia coli DNA polymerase III, are controlled by heat shock proteins

“…Both in vivo and in vitro experiments indicate that insertion of a base opposite a lesion is not rate limiting but that extension from the mispaired terminus is (6,18). The two modifications of Pol III that appear to be required are inhibition of the 3'-*5' exonuclease activity of epsilon to prevent the complex from stalling at the mispaired terminus (54) and improving the ability of the polymerase to extend from the mispaired terminus (2,6,18). A number of lines of evidence support the idea that RecA and UmuDC are involved in performing these alterations: (i) overproduction * Corresponding author.…”

“…Current theories of SOS mutagenesis postulate that mutations occur when Pol III replicates past DNA lesions. Since reconstituted Pol III holoenzyme is poor at synthesizing past lesions in vitro (18), it appears to have to be modified in vivo to allow translesion synthesis. Both in vivo and in vitro experiments indicate that insertion of a base opposite a lesion is not rate limiting but that extension from the mispaired terminus is (6,18).…”

“…Since reconstituted Pol III holoenzyme is poor at synthesizing past lesions in vitro (18), it appears to have to be modified in vivo to allow translesion synthesis. Both in vivo and in vitro experiments indicate that insertion of a base opposite a lesion is not rate limiting but that extension from the mispaired terminus is (6,18). The two modifications of Pol III that appear to be required are inhibition of the 3'-*5' exonuclease activity of epsilon to prevent the complex from stalling at the mispaired terminus (54) and improving the ability of the polymerase to extend from the mispaired terminus (2,6,18).…”