The active form of DNA polymerase V is UmuD'(2)C-RecA-ATP - PubMed (original) (raw)

The active form of DNA polymerase V is UmuD'(2)C-RecA-ATP

Qingfei Jiang et al. Nature. 2009.

Abstract

DNA-damage-induced SOS mutations arise when Escherichia coli DNA polymerase (pol) V, activated by a RecA nucleoprotein filament (RecA*), catalyses translesion DNA synthesis. Here we address two longstanding enigmatic aspects of SOS mutagenesis, the molecular composition of mutagenically active pol V and the role of RecA*. We show that RecA* transfers a single RecA-ATP stoichiometrically from its DNA 3'-end to free pol V (UmuD'(2)C) to form an active mutasome (pol V Mut) with the composition UmuD'(2)C-RecA-ATP. Pol V Mut catalyses TLS in the absence of RecA* and deactivates rapidly upon dissociation from DNA. Deactivation occurs more slowly in the absence of DNA synthesis, while retaining RecA-ATP in the complex. Reactivation of pol V Mut is triggered by replacement of RecA-ATP from RecA*. Thus, the principal role of RecA* in SOS mutagenesis is to transfer RecA-ATP to pol V, and thus generate active mutasomal complex for translesion synthesis.

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Figures

Figure 1

Figure 1. DNA synthesis by pol V Mut or pol V transactivated by RecA*

The p/t DNA is a hairpin containing a 3-nucleotide template overhang. a, b, DNA synthesis by pol V Mut–(RecA E38KΔC17) (a) or pol V Mut–(wild-type RecA) (b) in the absence of RecA*. c, d, DNA synthesis by pol V transactivated by RecA E38KΔC17* (c) or wild-type RecA* (d). e, f, DNA synthesis by pol V Mut–(RecA E38KΔC17) (e) or pol V Mut–(wild-type RecA) (f) undergoing deactivation as a function of time. g, Deactivation of pol V Mut–(RecA E38KΔC17) and pol V Mut–(wild-type RecA) measured by quantifying DNA synthesis obtained from data in panels e and f, respectively. Complete reactivation of deactivated pol V Mut is observed by addition of trans RecA* at each delay time. h, pol V Mut performs one round of DNA synthesis and cannot reinitiate synthesis on separate p/t DNA substrate. The concentration of p/t DNA is 1 μM. We estimate that the fraction of active polymerase in the reaction is 50% for pol V Mut–(RecA E38KΔC17) and 25% for pol V Mut–(wild-type RecA). i, Addition of trans RecA E38KΔC17* enables pol V Mut–(RecA E38KΔC17) to reinitiate DNA synthesis on a separate p/t DNA substrate. Cycling also occurs using pol V Mut–(wild-type RecA) in conjunction with trans wild-type RecA* (data not shown). Trans RecA* is at 1 μM when present.

Figure 2

Figure 2. Activated pol V Mut complex is composed of UmuD′2C–RecA–ATP

a, b, DNA synthesis by pol V Mut–(wild-type RecA) (b), but not pol V Mut–(RecA E38KΔC17) (a), requires the presence of ATPγS or ATP. c, d, Activated pol V Mut–(wild-type RecA) complex contains UmuC, UmuD′ and wild-type RecA, in a 1:1:1 ratio. e, Fl-wild-type RecA and ATPγS remain bound in deactivated pol V Mut–(Fl-wild-type RecA) complexes as measured by affinity chromatography. f, Reactivation of pol V Mut–(Fl-wild-type RecA) is achieved by replacing Fl-wild-type RecA and [35S]ATPγS bound to deactivated pol V Mut–(Fl-wild-type RecA) with unlabelled wild-type RecA and ATPγS from wild-type RecA*. Reactivated pol V Mut–(Fl-wild-type RecA) complexes were isolated as in panel e.

Figure 3

Figure 3. Determination of the molecular mass of pol V Mut–(wild-type RecA) by MALS

a, After wild-type-RecA*-mediated transactivation of UmuD′2C and removal of wild-type RecA*, the mixture of pol V Mut–(wild-type RecA) and non-activated pol V was resolved by size-exclusion chromatography (upper trace), and the molecular mass corresponding to each peak was measured by MALS. Non-activated pol V run separately on the silica gel elutes at 18.4 min (lower trace). b, Silver-stained SDS–polyacrylamide gel showing the protein composition from the two peaks contained in panel a (upper trace).

Figure 4

Figure 4. Pol V activation requires transfer of a RecA molecule from the DNA 3′ end of RecA*

a, The amount of RecA transferred to pol V from exposed 3′- or 5′-tips of RecA* are similar for wild-type RecA*, RecA E38KΔC17* and RecA F117S*. b, DNA synthesis catalysed by pol V Mut–(wild-type RecA) or pol V Mut–(RecA E38KΔC17) occurs when RecA is transferred from the 3′-RecA* tip (left gel). Pol V Mut cannot synthesize DNA when RecA transfer takes place from the 5′-RecA* tip (right gel). Mutagenically inactive RecA F117S* fails to activate pol V.

Figure 5

Figure 5. Model depicting pol V activation, deactivation and reactivation

Pol V (UmuD′2C) is barely active in the absence of RecA*. A molecule of RecA (red circle) and ATP (green triangle) is transferred from the DNA 3′-end of RecA* to form a mutasomal complex (pol V Mut) containing UmuD′2C–RecA–ATP; this catalyses TLS in the absence of RecA*. After TLS, pol V Mut undergoes rapid deactivation upon dissociation from DNA, with RecA and ATP retained in the complex. When pol V Mut is activated, we surmise that RecA–ATP is bound to UmuD′2, and that dissociation from p/t DNA triggers a repositioning of RecA–ATP to bind with UmuC to deactivate pol V Mut. Reactivation of pol V Mut involves a replacement of RecA–ATP from the RecA* 3′-tip. Free pol V Mut undergoes slow deactivation, with RecA and ATP retained in the complex. A damaged DNA base is shown as a black cross in the template strand.

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