Stalled transcription complexes promote DNA repair at a distance

Abstract

Transcription-coupled nucleotide excision repair (TCR) accelerates the removal of noncoding lesions from the template strand of active genes, and hence contributes to genome-wide variations in mutation frequency. Current models for TCR suppose that a lesion must cause RNA polymerase (RNAP) to stall if it is to be a substrate for accelerated repair. We have examined the substrate requirements for TCR using a system in which transcription stalling and damage location can be uncoupled. We show that Mfd-dependent TCR in bacteria involves the formation of a damage search complex that can detect lesions downstream of a stalled RNAP, and that the strand specificity of the accelerated repair pathway is independent of the requirement for a lesion to stall RNAP. We also show that an ops (operon polarity suppressor) transcription pause site, which causes backtracking of RNAP, can promote the repair of downstream lesions when those lesions do not themselves cause the polymerase to stall. Our findings indicate that the transcription-repair coupling factor Mfd, which is an ATP-dependent superfamily 2 helicase that binds to RNAP, continues to translocate along DNA after RNAP has been displaced until a lesion in the template strand is located. The discovery that pause sites can promote the repair of nonstalling lesions suggests that TCR pathways may play a wider role in modulating mutation frequencies in different parts of the genome than has previously been suspected.

Keywords: ATPase; DNA translocase; UvrA; protein roadblock.

Fig. 1.

Effect of stalling RNAP upstream of a bio-dT lesion. (A) DNA substrates. A single bio-dT was present in the template strand (B5-bio-TS) or nontemplate strand (B5-bio-NTS). (B) Effect of a template strand bio-dT lesion on in vitro transcription. pSRTB8B5 is unmodified supercoiled DNA. B5-bio-TS and B5-ctrl-TS are closed circular substrates into which a bio-dT–containing or unmodified control oligonucleotide had been ligated. Reactions contained 20 nM RNAP. The T7A1 transcript terminates 281 bp downstream of the transcription start site. The RNA I transcript is encoded as part of the ColE1 DNA replication origin of the pSR plasmids. The locations of the bio-dT and a product caused by pausing or termination within the A/T-rich TFO binding site (labeled #) are indicated. (C) Patch synthesis assay monitoring repair of bio-dT in the template strand and nontemplate strand. All reactions contained DNA polymerase, DNA ligase, ATP, NADH, and the radiolabeled dNTP mixture required for patch synthesis. “NER Repair mix” indicates that UvrA, UvrB, UvrC, and UvrD were added; “Free transcription” indicates that RNAP was added with GTP, CTP, and UTP; and “Stalled RNAP” indicates that RNAP was added with GTP, CTP, adenylyl (3′-5′) uridine (ApU), and 3′ dUTP. (D) Quantification of the [³²P]-dATP incorporated into the bio-dT–containing strand of B5-bio-TS and B5-bio-NTS during patch synthesis assays, normalized to the amount incorporated into the UV-irradiated pHWL1-2 control in each lane. Values are the average of at least three repeats and are shown with SD.

Fig. 2.

Effect of stalling an RNAP upstream of a CPD lesion. (A) DNA substrate. A single CPD was present in the template strand. (B) Patch synthesis assay monitoring repair of a single CPD in the template strand. Additions are as described for Fig. 1. (C) Quantification of the [³²P]-dATP incorporated into the template strand of B3-CPD-TS during patch synthesis assays, normalized to the amount incorporated into the UV-irradiated pHWL1-2 control in each lane. Values are the average of at least three repeats and are shown with SD.

Fig. 3.

Effect of a protein roadblock on TCR at a distance. (A) DNA substrate. Plasmid pHWL1-T7A1-2lacO contains a lac operator between the T7A1 promoter and a repair-reporter cassette. DNA lesions were introduced randomly into both strands of the plasmid by exposure to 30 J/m² of UV light. (B) Quantification of the [³²P]-dATP incorporated into the template and nontemplate strands of the pHWL1-T7A1-2lacO reporter cassette during patch synthesis assays normalized to the amount incorporated into the UV-irradiated pHWL1-2 control in each lane. Additions are as described for Fig. 1. Values are the average of at least three repeats and are shown with SD. A sample dataset is shown in Fig. S3.

Fig. 4.

DNA translocation by Mfd. (A) Effect of distance on efficiency of translocation by Mfd, measured by TFO-displacement assay. All substrates allow RNAP to be stalled at +21. “Gap” indicates the distance between +21 and the promoter-proximal end of the triplex when the TFO is bound to the substrate. Data labeled pSRTB1, pSRTB8, and pSRTB9 represent experiments in which RNAP was stalled and Mfd and dATP were then added to initiate DNA translocation. Control experiments in which RNAP, Mfd, or dATP was omitted were conducted on pSRTB9. (B) Effect of a Lac repressor roadblock on TFO displacement using substrates that contain (pSRTBlacO) or lack (pSRTB8) a lac operator between the RNAP stall site and the TFO binding site. RNAP was stalled in the presence or absence of Lac repressor, and Mfd and dATP were then added to initiate DNA translocation. (C) Effect of lesions in the template strand between the RNAP stall site and the TFO binding site. The DNA between the stall site and the TFO binding site was either undamaged (B3-ctrl-TS) or contained a single bio-dT (B3-bio-TS) or CPD (B3-CPD-TS) in the template strand. RNAP was stalled, and Mfd and dATP were then added to initiate DNA translocation. Values in A and C are the average of at least three repeats and are shown with SD. Values in B are the average of two or three repeats and are shown with range.

Fig. 5.

Effect of pausing RNAP upstream of a nonstalling lesion. (A) DNA substrates. (B) Patch synthesis assay monitoring repair of a bio-dT in the template strand in a substrate containing an ops pause site (B3ops-bio-TS) or a control lacking the ops pause site (B5-bio-TS). Additions are as described for Fig. 1. (C) Quantification of the [³²P]-dATP incorporated into the template strand of each substrate during patch synthesis assays normalized to the amount incorporated into the UV-irradiated pHWL1-2 control in each lane. Values are the average of at least three repeats and are shown with SD.