Ultraviolet photoproducts at the ochre suppressor mutation site in the gln U gene of Escherichia coli: Relevance to “mutation frequency decline”

Abstract

Ochre suppressor mutations induced by UV in the Escherichia coli glnU tRNA gene are CG to TA transitions at the first letter of the anticodon-encoding triplet, CAA. Premutational UV photoproducts at this site have long been known to exhibit an excision repair anomaly ("mutation frequency decline" or MFD), whereby postirradiation inhibition of protein synthesis enhances their excision and reduces suppressor mutation yields ten-fold. We sought to clarify the basis of this unique repair response by determining the spectrum of UV photoproducts on both strands of a 36 bp region of glnU which includes the anticodon-encoding triplet. We found that four different photolesions are produced within the 3 bp sequence corresponding to the tRNA anticodon: (i) on the transcribed strand, TC (6-4) photoproducts and TC cyclobutane dimers are formed in equal numbers at the site of the C to T transition, indicating that this site is a hotspot for the usually less frequent (6-4) photoproduct; (ii) on the nontranscribed strand, TT dimers are found opposite the second and third letters of the anticodon-encoding triplet, adjacent to the mutation site; and (iii) on the nontranscribed strand, an alkali-sensitive lesion other than a (6-4) photoproduct is formed, apparently at the G in the mutation site. We suggest that mutation frequency decline may reflect excision repair activity at closely spaced UV lesions on opposite strands, resulting in double-strand breaks and the death of potential mutants.

Roots: Mutation frequency decline revisited

“…Consistent with this was our later finding that TC cyclobutane dimers are abundantly fonned in glnU at this site, which is also a hotspot for the fonnation of TC(6,4) photoproducts (24).…”

“…Because MFD-promoting conditions lead to repression of tRNA genes (the 'stringent response'), one possibility was that the repressed state of these genes might enhance the exciseability of the target lesion (21). Later, however, Engstrom and Bockrath showed that MFD occurs in a re1 mutant, which presumably does not terminate transcription of tRNA genes under conditions that that would otherwise evoke the stringent r e~p o n s e (~~) , and Garvey et al (24) confinned this result in a rel-deleted strain.…”

“…Furthermore, early indications of the SOS response were beginning to compel my attention, and soon became my major focus. Except for detailed characterization of the Mfd-phenotype(2ss26), and, much later, analysis of the distribution of UV photoproducts in the critica1 region of the cloned glnU gene (24), work in my laboratory no longer centered on MFD.…”

Escherichia coli mfd mutant deficient in "mutation frequency decline" lacks strand-specific repair: in vitro complementation with purified coupling factor.

“…MFD is a highly specialized repair response not known to affect any premutational lesions in DNA except those located at the site corresponding to the first letter of the anticodon on the template strand of tRNA genes glnU (ochre) and glnV (amber) (16,17,23), a region rich in potential secondary structure. Even if the effect of TRCF is to stimulate repair on the template strand as a whole, it may not do so at that specific location because of a unique configuration assumed by the anticodon region during protein synthesis.…”

Mechanisms of transcription-repair coupling and mutation frequency decline.

“…One class, exemplified by uvr mutants, was deficient in excision repair and was UV sensitive; the second class was defined by a single gene, mfd. Further studies of the mfd mutant provided some clues for how this gene might function (26)(27)(28): (i) the mfd mutant was capable of excision repair; (ii) Mfd-cells were only slightly more sensitive to UV than Mfd+ cells; (iii) Mfdcells had a normal spontaneous mutation rate; (iv) Mfd-cells had a fivefold higher UV-induced mutation rate than wild-type cells at all loci tested; (v) Mfd-cells removed pyrimidine Representation of a transcription bubble. ++++, transient positive supercoiling preceding, and ----, transient negative supercoiling following, the transcribing RNAP.…”