Transcriptional pausing to scout ahead for DNA damage

Abstract

A cell's genome is under constant threat of damage, which if not repaired can lead to mutations or cell death. Common forms of DNA damage found in nature include cyclobutane pyrimidine dimers and 6-4 photoproducts induced by UV-irradiation. These and other helix-distorting lesions are removed by a highly conserved process called nucleotide excision repair (NER) that is found in every kingdom of life (1). NER is initiated in two general ways: by damage recognition proteins that survey the entire genome for damage or lesion-induced transcriptional stalling. This latter pathway, called transcription-coupled repair (TCR), first reported in mammalian cells and then in bacteria, is initiated when RNA polymerase (RNAP) is arrested at a DNA lesion embedded in the transcribed strand (2, 3). However, before DNA repair enzymes obtain access, the stalled RNAP must be pushed away from the lesion by the action of DNA translocases. Thus, the repair "coupling factors," which recognize the stalled RNAP, must work to both displace the polymerase and simultaneously enlist the repair proteins to remove the damage. In bacteria, two different TCR pathways have emerged involving two different DNA helicases, which help to displace RNAP. The Mfd (mutation frequency decline) protein, also called transcription-repair coupling factor, uses its helicase fold and ATP hydrolysis to literally push RNAP forward (downstream) past the damaged site (Fig. 1A) (reviewed in ref. 4), whereas in a newly discovered alternative pathway UvrD (helicase II) tows the RNAP backward (upstream) with the help of the transcription elongation factor, NusA (5). This second approach more closely resembles what is thought to occur in mammalian cells during TCR (6). As described below, Mfd targets the nucleotide excision repair system to sites of damage through its direct interaction with a stalled RNAP. However, nature has gone even further in devising ways to find and remove potentially RNAP-blocking DNA damage. As described by Haines et al. (7) in PNAS, Nigel Savery's group at the University of Bristol have found that the Mfd protein that normally accompanies the translocating RNAP can be sent ahead of a blocked RNAP to scout for damage in the transcribed strand and facilitate the recruitment of the bacterial NER machinery.During the past 15 y structural biologists and biochemists have described in wonderful detail how bacterial NER proteins process and remove DNA damage (reviewed in ref. 1). Once the stalled RNAP has been removed from the damage site, Mfd or UvrD are thought to recruit the UvrAB complex (made up of a UvrA dimer and one or two UvrB molecules) to the site of the damage. Mfd is a multidomain protein that shares a protein fold with UvrB and this motif is responsible for the interaction face with UvrA. Mfd undergoes a large conformational change during its handling of stalled RNAP. This process exposes Mfd's UvrB homology domain, which is believed to attract UvrA to the damaged site. Single-molecule studies have shown that the UvrAB complex searches fo...

De novo assembly of a chromosome-level reference genome of the ornamental butterfly Sericinus montelus based on nanopore sequencing and Hi-C analysis

“…The directional alterations in the metabolism of some amino acids may impact the efficacy of promoting tolerance to AAs ( Stepka et al, 2021 ). For example, the eukaryotic transcription process mediated by RNA polymerase II (pol II) may be paused by several situations such as DNA damage ( Van Houten and Kisker, 2014 ). In this case, the elongin complex can mediate the ubiquitylation and degradation of the largest subunit of pol II (Rpb1) to stimulate transcription elongation ( Yasukawa et al, 2008 ).…”

Analysis of the nucleotide content of Escherichia coli promoter sequences related to the alternative sigma factors

“…Ongoing studies relate the interaction between the presence of the G nucleotide in this region and pausing mechanisms to scout ahead for DNA damage. As related by Van Houten and Kisker, a Mutation Frequency Decline protein uses its helicase fold and ATP hydrolysis in order to push the RNAp enzyme downstream, past the damaged site. An alternative pathway involves the work‐in‐group of a protein called UvrD and a transcription elongation factor (NusA) that is capable of towing the RNAp backwards.…”

“…Each σ-dependent promoter sequence presents different conserved motifs, which distinguish themselves from one another. 1 In total, six distinct alternative σ factors are known in E. coli, for instance: σ 19 , σ 24 , σ 28 , σ 32 , σ 38 , and σ 54 .…”

A tale of two cities: A tribute to Aziz Sancar's Nobel Prize in Chemistry for his molecular characterization of NER

“…It was later shown by several groups who helped solve the structure and function of TRCF/MFD that this protein shares a UvrB-homology domain to which UvrA binds [68–70]. It is believed that once RNA polymerase stalls at a damaged site, binding of MFD triggers opening of this UvrA interacting domain to facilitate pushing RNA polymerase off the damaged site through its translocase activity while simultaneously recruiting UvrA to the site [71–75]. …”