Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli

Abstract

DNA polymerase V, composed of a heterotrimer of the DNA damage-inducible UmuC and UmuD(2)(') proteins, working in conjunction with RecA, single-stranded DNA (ssDNA)-binding protein (SSB), beta sliding clamp, and gamma clamp loading complex, are responsible for most SOS lesion-targeted mutations in Escherichia coli, by catalyzing translesion synthesis (TLS). DNA polymerase II, the product of the damage-inducible polB (dinA ) gene plays a pivotal role in replication-restart, a process that bypasses DNA damage in an error-free manner. Replication-restart takes place almost immediately after the DNA is damaged (approximately 2 min post-UV irradiation), whereas TLS occurs after pol V is induced approximately 50 min later. We discuss recent data for pol V-catalyzed TLS and pol II-catalyzed replication-restart. Specific roles during TLS for pol V and each of its accessory factors have been recently determined. Although the precise molecular mechanism of pol II-dependent replication-restart remains to be elucidated, it has recently been shown to operate in conjunction with RecFOR and PriA proteins.

Figure 1

Primer-template DNA constructs used to reconstitute SOS mutagenesis in vitro. (a) When pol V, incubated in the presence of RecA, SSB, β sliding clamp, and γ clamp loading complex is used to copy a 30-mer/M13 primer-template DNA, synthesis proceeds past the lesion X to the end of the template strand (18). TLS requires the presence of pol V and RecA proteins for the case of TT cis-syn photodimers, TT (–4) photoproducts, and abasic sites (19). (b) RecA, SSB, and pol V bind to regions of ssDNA far from the lesion site. The use of a shorter p/t DNA (30-mer/240-mer) reduces nonessential ssDNA, enabling a measurement of the stoichiometries and the effects of each mutasomal component on TLS (26).

Figure 2

A “cowcatcher” model describing DNA polymerase V mutasome-catalyzed TLS. (a) A DNA polymerase III holoenzyme replication complex (HE; yellow) stalls when encountering a template lesion (X). The continued unwinding action of DnaB helicase (not shown) opens up a stretch of ssDNA template downstream of X. (b) pol III core dissociates from the 3′-primer end proximal to the lesion, and an activated RecA nucleoprotein filament (RecA*; light blue) is assembled in a 5′ to 3′ direction on the ssDNA in a reaction requiring ATP, but not ATP hydrolysis. (c) The RecA nucleoprotein filament continues to advance to reach the site of DNA damage. (d) pol V binds to the 3′-primer end vacated by pol III core. The activity and binding affinity of pol V are strongly stimulated by the presence of RecA, SSB, and β sliding clamp (12, 26). (e) The key feature of the model is that pol V (red) + SSB (green), operating jointly as a locomotive cowcatcher, strip RecA from the DNA template in a 3′ to 5′ direction immediately ahead of an advancing pol V molecule. The cowcatcher stripping reaction does not require ATP hydrolysis and takes place concurrently with the “standard” 5′ to 3′ RecA filament disassembly reaction requiring ATP hydrolysis. (f) After TLS, pol V dissociates from DNA when contact with the tip of a RecA filament is lost. Thus, bidirectional RecA filament disassembly helps to confine mutations to DNA damage sites by ensuring that undamaged DNA template bases are not copied by the low fidelity pol V. The pol III HE replication complex reassembles after dissociation of pol V. The sketch in e has been enlarged relative to the other parts of the figure to emphasize the cowcatcher aspects of the TLS model. Data supporting this model are contained in ref. .

Figure 3

Sketch of a nuclease protection analysis used to demonstrate the 3′ → 5′ disassembly of a RecA nucleoprotein filament by pol V + SSB. (a) A “stabilized” RecA nucleoprotein filament (blue circles) has been formed with ATPγS. The RecA filament cannot disassemble in the absence of ATP hydrolysis and is therefore refractory to cleavage by HinfI and by λ 5′ → 3′ double-stranded or RecJ 5′ → 3′ single-stranded exonucleases. Blocked cleavage reactions are depicted by crossed-out arrows. A <1-min reaction with pol V Mut is sufficient to incorporate C opposite G and to extend the primer past the template lesion (X); however, the extended primer does not yet reach the HinfI restriction site located 10 nt downstream from X. Therefore, a HinfI restriction enzyme fails to cut the DNA. (b) A 3-min reaction with pol V Mut is sufficient to copy past the HinfI restriction site, enabling cleavage by the restriction enzyme and subsequent digestion of the ³²P-labeled template strand using a combination of RecJ + λ exonucleases (bottom portion of sketch). When the HinfI restriction enzyme is omitted from the reaction, digestion of the template strand does not occur because pol V Mut has not yet copied to the 5′-end of the template strand (top portion of sketch). (c) A 10-min reaction with pol V Mut is sufficient to reach the end of the template strand, enabling digestion with λ exonuclease. The experimental data documenting the pol V + SSB-catalyzed RecA stripping reaction are contained in ref. .