Transcription exposes ssDNA.
The most common base substitution events in the spectra of background mutations in
E. coli and mammalian cells are G · C-to-A · T transitions. Fix and Glickman (
28) observe that 77% of these mutations originate on the nontranscribed strand in
E. coli mutants unable to repair deaminated cytosines. This suggests that the unprotected single strand in the transcription “bubble” is significantly more vulnerable to mutations than the transcribed strand, which is protected as a DNA-RNA hybrid (Fig.
1A). The frequency of UV-induced lesions in the
lacI gene is also higher in the nontranscribed strand than in the transcribed strand (
46). In fact, cytosines deaminate to uracils in ssDNA at more than 100 times the rate in dsDNA (
31,
32,
60). The relative mutability of the nontranscribed strand is also seen in a plasmid system in which a fourfold increase in the frequency of transitions occurs selectively in the nontranscribed strand when transcription is induced (
4). Transcription may therefore be a prerequisite for many C-to-T transition mutations, since other mechanisms resulting in the transient generation of single-stranded sequences, such as replication or breathing (
102) do not lead to asymmetry in the two strands. Apparently, the observed strand bias cannot be explained by transcription-coupled repair (
36), since base mismatches are poor substrates for this kind of repair, and the same strand bias is observed when the host is deficient in repairing U · G and T · G mismatches (
4). Thus, transcription may be implicated as a major cause of background transition mutations in nature.
Transcriptional activation as a mechanism for increasing mutation rates was first proposed in 1971, by Brock (
8) and Herman and Dworkin (
38). Their work demonstrates that
recA-independent
lac reversion rates of frameshift and point mutations are higher when transcription is induced by isopropyl-β-
d-thiogalactopyranoside (IPTG), and that the effect is specific. More recently, specifically induced, transcription-enhanced mutations have also been shown for a
lys frameshift mutation in
Saccharomyces cerevisiae (
16,
74). Starvation-induced stringent response mutations in
E. coli (
62,
109-111) and
Bacillus subtilis (
90) occur as a result of transcriptional activation triggered by gene derepression, not induction. In this system, mutations arise during the transition between growth and stationary phase and they are
recAindependent, similar to the
lac reversions mentioned above. This distinguishes them from prolonged stress-induced adaptive mutations (
11) and from DNA damage-induced SOS mutagenesis (
104), both of which require
recA (and will not be discussed in this minireview). It is noteworthy that the experiments described above on the effects of artificially induced transcription on mutation rates in growing cells are all examples of specifically directed mutations. However, none of the researchers come to that conclusion or challenge the assumptions and implications inherent in the experiments of Luria and Delbruck (
63), which reinforce neo-Darwinism. This situation may be due to the dominance of current dogma and to the assumption that mechanisms operative during growth cannot also be critical during evolution under conditions of environmental stress. In fact, the limited evidence now available suggests that only growing cells, or cells in transition between growth and stationary phase, have the metabolic potential required for specific, transcription-induced mutations in response to environmental challenge. Thus, IPTG induction enhances
lac reversion rates in growing cells (
38) but not in cells subjected to prolonged stress (
17). Transcriptional activation is the mechanism for enhancing mutation rates both in the artificially induced systems and in stringent response mutations (
90,
111; J. M. Reimers, A. Longacre, and B. E. Wright, Conf. DNA Repair Mutag., 1999). However, only the latter are relevant to evolution, since they occur naturally as a result of starvation-induced derepression. Mutations that most benefit organisms and accelerate evolution may occur as an immediate response to imminent starvation, when cells still have the metabolic resources to respond specifically to the particular conditions of stress at hand.
Transcription drives localized supercoiling.
Chromosomal DNA from bacterial cells is negatively supercoiled. The level of global negative supercoiling in
E. coli cells is maintained within a physiologically acceptable range by two opposing enzyme activities: DNA gyrase, which introduces negative supercoils, and topoisomerase I, which relaxes them. Investigations with plasmids grown in
E. coli (
59,
81) demonstrate the presence of stem-loop structures in naturally occurring supercoiled circular DNA molecules (Fig.
1B). Analyses with single strand-specific nuclease show that DNA molecules with high superhelical densities are selectively cleaved, in contrast to their linearized counterparts with which they are in dynamic equilibrium in vivo. The sequence surrounding the area of cleavage reveals inverted complementary sequences that hydrogen bond to become the stem separated by noncomplementary bases that become the single-stranded loop and the substrate for nuclease cleavage. Such complex structures form preferentially in easily denatured AT-rich stretches of DNA and occur about 10,000 times more frequently than expected by chance (
30,
59), suggesting their selection during evolution. Data indicate that stem-loop-based recombination may have evolved in the early “RNA world” (
94) and that the potential to generate stem-loops was later conserved, for example, in hypervariable snake venom genes under strong selection to keep one step ahead of both predators and prey (
29).
A number of variables, such as temperature, anaerobiosis, osmolarity, and nutritional shifts, affect DNA supercoiling (
1,
19,
47,
85,
86). Some environmental perturbations affect plasmid systems and chromosomes in a similar manner, while some apparently do not (
25). Transcription both responds to and promotes changes in supercoiling. The optimal level of supercoiling for gene expression varies for different genes, and supercoiling-induced conformational changes may be required for structural changes in regulatory complexes and for recognition by RNA polymerase (RNAP) (
85). Transcription has a profound effect on supercoiling, because RNAP distorts and destabilizes dsDNA. As indicated in the twin-domain model (Fig.
1B) of Liu and Wang (
61), negative supercoiling is generated behind, and positive supercoiling in front of, the advancing RNAP transcription complex. Many investigations provide evidence demonstrating that transcription drives supercoiling in vivo (
1,
19,
20,
27,
86) and that the wave generated can be as long as 800 bp (
47). Negative supercoiling induces and stabilizes a transition from the right-handed B-form to the left-handed Z-form of DNA (
42); a chemical assay detecting these distortions reveals that transcription-induced supercoiling is highly localized (
47,
86). During the induction of transcription, supercoils are found inside each transcribed region, as well as upstream and downstream of each individual RNAP complex. Transcription from a strong promoter leads to greater negative supercoiling than transcription from a weak one (
27). A major role of DNA topoisomerase I is now considered to be the relaxation of local negative supercoiling during transcription, thus preventing unacceptably high levels of supercoiling and associated R-loops that form when nascent RNA moves behind the advancing RNAP to bond with its original template DNA (
69,
70,
105). The bulk of plasmid DNA does not exhibit stem-loop conformations during logarithmic growth. However, supercoiling may play a particularly important role in stressed cells, in which a disruption can occur between transcription and translation, thus promoting both R-loop formation and supercoiling (
68,
69). The inhibition of protein synthesis by chloramphenicol, which uncouples transcription and translation, induces stem-loop formation in the overwhelming majority of DNA molecules (
19).
When
E. coli is grown with limiting levels of glucose (Fig.
2), a burst in supercoiling occurs precisely at the moment of glucose depletion, as the cells cease logarithmic growth and enter stationary phase (
1). This is also the moment at which a sharp increase occurs in the concentration of cyclic AMP (cAMP) (
10), ppGpp (
23,
51), ς
S (
50), and about 30 new proteins, including β-galactosidase (
34,
37). The increase in negative supercoiling under these circumstances can be due to the increase in transcription known to occur as a result of derepression in response to starvation for any essential nutrient. The two “alarmones,” cAMP and ppGpp, activate transcription in derepressed genes in a number of systems. Both are required for the synthesis of enzymes that catabolize alternative carbon sources, such as β-galactosidase (
83,
84,
91). The alarmone ppGpp activates the synthesis of ς
S (
33), which in turn governs the expression of a number of stationary-phase genes involved in the starvation-mediated resistance to osmotic, oxidative, and heat damage (
37,
72). Under circumstances in which a group of related genes become activated, such as those dependent upon ς
S, the topological changes in DNA could provide a mechanism by which transcriptional activation in one gene may influence adjacent genes (
47,
85). Changes in stem-loop formation and superhelicity similar to that caused by glucose starvation (Fig.
2) are also observed immediately following amino acid starvation or the inhibition of protein synthesis (
19). Within 30 min following treatment with chloramphenicol or valine (which creates an isoleucine deficit and ppGpp accumulation in
E. coli), stem-loop formation is evident. Isoleucine starvation results in the formation of stem-loops in a much smaller number of DNA molecules than are affected by chloramphenicol inhibition, consistent with the selective derepression of relatively few genes in the absence of a single amino acid.
In response to starvation for any essential metabolite, the immediate problem is addressed specifically (e.g., derepression of a higher-affinity transport system for that metabolite), coupled with a general increase in stress resistance. Starvation results in derepression, and transcription drives localized supercoiling; the formation of stem-loop structures at regions of high superhelicity results in localized hypermutation (Fig.
1). Although energetic considerations do not favor the creation of complex structures in metabolically inactive dsDNA, transcription clearly accelerates supercoiling and transitions to secondary DNA structures (
1,
19,
20,
27,
47,
59,
69,
70,
81,
86).