Nucleotide excision repair (NER) machinery recruitment by the transcription-repair coupling factor involves unmasking of a conserved intramolecular interface

Abstract

Transcription-coupled DNA repair targets DNA lesions that block progression of elongating RNA polymerases. In bacteria, the transcription-repair coupling factor (TRCF; also known as Mfd) SF2 ATPase recognizes RNA polymerase stalled at a site of DNA damage, removes the enzyme from the DNA, and recruits the Uvr(A)BC nucleotide excision repair machinery via UvrA binding. Previous studies of TRCF revealed a molecular architecture incompatible with UvrA binding, leaving its recruitment mechanism unclear. Here, we examine the UvrA recognition determinants of TRCF using X-ray crystallography of a core TRCF-UvrA complex and probe the conformational flexibility of TRCF in the absence and presence of nucleotides using small-angle X-ray scattering. We demonstrate that the C-terminal domain of TRCF is inhibitory for UvrA binding, but not RNA polymerase release, and show that nucleotide binding induces concerted multidomain motions. Our studies suggest that autoinhibition of UvrA binding in TRCF may be relieved only upon engaging the DNA damage.cysteine cross-linking | transcription | ATPase stimulation | UvrB R NA polymerase (RNAP) stalled at DNA lesions on the transcribed strand elicits a preferential pathway for nucleotide excision repair (NER) called transcription-coupled repair (TCR), which is present in Bacteria and Eukarya (1). Bacterial transcription-repair coupling factor (TRCF; also known as Mfd) orchestrates this process by specific recognition of the transcription and NER assemblies, which reflects its twofold role. First, TRCF relieves transcription-dependent NER inhibition due to occlusion of the DNA lesion by RNAP (2). TRCF, an SF2 ATPase with dsDNA translocase but no helicase activity (3), approaches the stalled RNAP from behind and induces its forward translocation by stepping on dsDNA using ATP hydrolysis (4, 5). The consequent collapse of the upstream end of the transcription bubble leads to massive destabilization of the otherwise stable ternary elongation complex (TEC) and transcription termination (4-7). Rho, the only other known bacterial enzymatic terminator, induces termination by a similar forward-translocation mechanism, but translocates along the nascent RNA (8). Second, TRCF recruits the Uvr(A)BC endonuclease to the unmasked lesion by binding to UvrA (4, 9). This initiates a cascade of events resulting in lesion excision and gap filling (4, 10). TRCF also has roles beyond TCR-in the rescue of replication forks stalled by head-on collisions with RNAPs (11), in the development of antibiotic resistance (12, 13), recombination (14, 15), and transcriptional regulation (16, 17).The crystal structure of apo TRCF (18) revealed a multimodular enzyme with eight domains connected by flexible linkers (Fig. 1A), an architecture that appears primed for large conformational changes, which are believed to be critical for coupling RNAP recognition to recruitment of NER enzymes. Domains D1 and D2 of TRCF are similar to the NER protein UvrB, which also binds UvrA (18,19), suggesting that these domains serve as a pl...

Mfd as a central partner of transcription coupled repair

“…The first catalytic step, which is relatively slow, could be explained by some major conformational changes of Mfd, dependent on ATP, allowing the unmasking of UvrA binding surface at the level of the Mfd D2-D7 domain (consistent with the recent SAXS -Small-angle X-ray scattering -results). 36,37 This idea is also supported by the observations that (i) a second RNA polymerase could only bind the promoter when the first RNA polymerase is trapped in this long-lived polymerases, 24 are not able to displace a DNA-lesion-dependent stalled-RNA polymerase. 25,26 Second, Mfd recruits UvrAB, allowing active DNA-lesion repair.…”

UvrA expression of Lactococcus lactis NZ9000 improve multiple stresses tolerance and fermentation of lactic acid against salt stress

“…Cultures were subjected to osmotic stress (15% NaCl), acid stress (pH 4.0), cold stress (-20°C) and Heat stress (46°C) for various times and the survival rate was determined thus initiating dissociation of the RNA polymerase ternary elongation complex. The rate of DNA repair depends on many factors, including the cell type, age of the cell, and extracellular environmental conditions (Deaconescu et al 2012). Temperature changes directly affect the growth characteristics of Lactobacillus delbrueckii and lead to DNA damage.…”

Stalled transcription complexes promote DNA repair at a distance

“…All of the proteins that are required for global NER are also required for TCR, and the steps subsequent to UvrA dissociation are thought to be common to the two pathways (9). Mfd and UvrB both interact with the same surface of UvrA, and the interaction between Mfd and UvrA involves a region of Mfd that is structurally homologous to part of UvrB (12,13). Mutational analysis of UvrA has shown that the ability of UvrA to distinguish between damaged and undamaged DNA is less important during TCR than during global NER (12), but the mechanism by which the Mfd-UvrA interaction accelerates repair is not understood.…”

“…Mfd also interacts with the UvrA protein, which is a component of the global NER apparatus (9,12,13). During global NER, damage is detected by a search complex made up of a UvrA dimer and two molecules of the repair protein UvrB.…”

“…stable binding of UvrB at the site of DNA damage, such a model would help to explain the decreased requirement for damage recognition by UvrA during TCR. Models have been proposed in which UvrA is recruited to Mfd shortly after Mfd binds to RNAP (12) or in which UvrA is not recruited until after Mfd itself has detected the lesion (13). Because Mfd and UvrB interact with UvrA in a similar fashion, they may compete with each other for UvrA, and it is possible that the damage search mechanism of TCR involves a Mfd/UvrA 2 /UvrB complex in place of the UvrB/UvrA 2 / UvrB complex involved in global NER.…”