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. 2012 Apr 13;46(1):18-29.
doi: 10.1016/j.molcel.2012.02.006. Epub 2012 Mar 8.

Mechanism of Translesion Transcription by RNA Polymerase II and Its Role in Cellular Resistance to DNA Damage

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Free PMC article

Mechanism of Translesion Transcription by RNA Polymerase II and Its Role in Cellular Resistance to DNA Damage

Celine Walmacq et al. Mol Cell. .
Free PMC article

Abstract

UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by RNA polymerase II (Pol II). If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair. Translesion synthesis depends on the trigger loop and bridge helix, the two flexible regions of the Pol II subunit Rpb1 that participate in substrate binding, catalysis, and translocation. Substitutions in Rpb1 that promote lesion bypass in vitro increase UV resistance in vivo, and substitutions that inhibit lesion bypass decrease cell survival after UV irradiation. Thus, translesion transcription becomes essential for cell survival upon accumulation of the unrepaired CPD lesions in genomic DNA.

Figures

Figure 1
Figure 1. Point mutations in Pol II affect cell tolerance to UV irradiation and CPD bypass in vitro
A. TEC structure of S. cerevisiae Pol II with the closed (cyan) and the open TL (yellow) [PDB: 2E2H (Wang et al., 2006) and PDB: 1Y1V (Kettenberger et al., 2003)] aligned using the VMD software (Humphrey et al., 1996). The opened TL conformation (in PDB: 1Y1V) structure required the TFIIS protein (not shown). The DNA backbone and RNA are shown in light and dark grey, respectively. Rpb1-E1103 is located at the base of the TL. Rpb1-G730 locates in the α21 helix, the outer region of the secondary channel (Cramer et al., 2001). Cartoon in the box shows contacts of the NTP with the closed TL and α20-α23 helices domain in the secondary channel. B. rpb1-E1103G mutation increases UV resistance of yeast while rpb1-G730D mutation exacerbates the UV sensitivity. Survival was averaged from at least 3–5 independent experiments. C. WT, E1103G or G730D TEC12 obtained on TT dimer-containing template (TS71-CPD38/NTS71-38) was incubated with NTPs for various times (see Experimental Procedures).
Figure 2
Figure 2. A CPD lesion stabilizes the TEC in the post-translocated states
A. Rear-end footprint experimental setup. The figure illustrates the predicted translocation register of lesion-stalled TECs. The length of the template DNA strand protected from Exo III digestion differs by one nucleotide between the backtracked (42-nt), the pre-translocated (41-nt) and the post-translocated (40-nt) state of TEC12. B. Dynamics of DNA degradation by Exo III revealed equilibrium between pre- and post-translocated states of the TEC. C. The lesion confers a shift of the equilibrium toward the post-translocated state but inhibits stabilization of the post-translocated state of Pol II by an incoming NTP. The quantification was based on the results of at least 3 separate experiments. D. Front-end footprint experimental setup. E. Mapping of the translocation register of Pol II front-end boundary revealed that the lesion does not promote backtracking of TEC12. F. The bottom panel depicts the distribution of the three distinct front-end boundaries detected in TEC12 at various incubation times with Exo III.
Figure 3
Figure 3. Pol II employs the A-rule to conduct ATP insertion opposite the 3’T-CPD
A. Preferential AMP incorporation opposite the 3’T-CPD. TEC12 was obtained from TEC9 on CPD-DNA as described in Fig. 1C and incubated with 1 mM individual NTP or 4 NTPs for different time. Individual NTP reactions were subsequently chased with 4NTPs for 15 min (C). Black and white arrows indicate normal incorporation products and misincorporation, respectively. The longer RNA A15 derived from AMP incorporation opposite both thymines followed by misincorporation of A for G. B. Non-templated AMP incorporation opposite an abasic site (3’aB) C. Apparent rate constants of the correct and incorrect NTP incorporation on regular, CPD and aB DNA.
Figure 4
Figure 4. UTP misincorporation opposite the 5’T-CPD abrogates lesion bypass
A. TEC13A obtained from TEC12 was incubated with 1 mM individual NTP for 120 min or 4 NTPs for indicated time. Individual NTP reactions were subsequently chased with 4 NTPs. AMP incorporation opposite the 5’T is required for lesion bypass while UMP misincorporation results in irreversible stalling. B. Rear-end Exo III footprinting of TEC13A obtained from TEC12 as described in Fig. 2B.
Figure 5
Figure 5. Crystal structure of TEC stalled at the CPD reveals foundation for the A-rule
A. A wire-frame representation of the crystal structure of the CPD lesion-containing TEC9 stalled upstream the CPD (positions i+1/i+2). Pol II TECs structures with a CPD lesion located in the downstream DNA (positions i+2/i+3) and within the active center (positions i/i+1) are also shown (Brueckner et al., 2007). The RNA, template and non-template DNA are highlighted in red, dark blue and cyan colors, respectively. The bridge helix (BH) is shown in green. The cartoons on the bottom highlight the details of the structure on the TT regular (right) and CPD-containing DNA (left). B. The details of the configuration of the RNA 3' end in the regular post-translocated TEC (PDB: 2E2H) and in its CPD-stalled counterpart from panel A. The numbers represent the distances in angstroms between the 3' purine base and the complementary pyrimidine in the template DNA. The structures on the right display a conformation of the BH and the i+1/i+2 DNA bases in the regular post-translocated TEC with the incoming NTP (I), the CPD-TEC without NTP (II), and in the CPD-TEC with the modeled purine nucleotide in the active center (the NTP coordinates taken from PDB: 2E2H). C. Pol II activity modulates UV resistance by RAD26-dependent and RAD26-independent pathways. Yeast strains carrying RPB1WT (square), rpb1-E1103G (circle) or rpb1-G730D (triangle) mutation in the rad16rad26 and rad26 background were irradiated with increasing UV doses. UV sensitivity of yeast cells in the isogenic rad16 background was also measured independently from Fig.1B (see Supplementary Text).
Figure 6
Figure 6. Steps in the CPD bypass
A. Individual steps in negotiation of Pol II with a CPD lesion (orange) and putative conformational changes in the TL (cyan) and the BH (green) leading to the error-free bypass or irreversible stalling. The NTP binding site (i+1 site) is shown in light green. An interaction between Rpb1-E1103 (yellow) and T1095 (green) residues of the TL during the lesion bypass is shown. G730D mutation (magenta) affects the proper alignment of the 4-helix α20-α23 domain (cyan cylinders) of Rpb1 subunit in the secondary channel with the incoming NTP (red). B. The proposed connection between GG-NER, TC-NER and TLS pathways in cellular resistance to UV-induced DNA damage. Encounter with a CPD lesion results in Pol II stalling at the damage site (red 'X'). The numbers mark: (1) Rad26-independent TLS followed by repair by GG-NER (2) initiation of TC-NER by Rad26 (in purple) recruitment to the CPD-stalled Pol II followed by (2a) the induction of TLS to expose the CPD to TC-NER, (2b) GG-NER, or resumption of transcription without repair (not shown). The dashed lane represents a pathway alternative to TLS, which involves Pol II ubiquitination and degradation after recruitment of Rad26/Def1 complex. TLS may contribute to UV cellular resistance by preventing collisions between both replication and transcription machineries. E1103G and G730D Pol II mutations promote and inhibit (1) and (2) TLS, respectively.

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