Genetic control of AAF-induced mutagenesis at alternating GC sequences: An additional role for RecA

Abstract

In a previous study, the forward mutation spectrum induced by the chemical carcinogen N-acetoxy-N-2-acetylaminofluorene was determined (Koffel-Schwartz et al. 1984). It was found that 90% of the induced mutations are frameshift mutations located within specific sequences (mutation hot spots). Two classes of mutation hot spots were found: (i) -1 frameshift mutations occurring within runs of guanines (i.e. GGGG----GGG; (ii) -2 frameshift mutations occurring within the NarI recognition sequence (GGCGCC----GGCC). In the present work, we further investigate the genetic requirements of these frameshift events by using specific reversion assays. Like UV-induced mutagenesis, frameshift mutations occurring within runs of G's (also referred to as the "slippage pathway") require the activated form of the RecA protein (RecA*). On the other hand, frameshift mutations occurring at the NarI site (the "NarI mutation pathway") require a LexA-controlled function(s) that is not UmuDC. The LexA-controlled gene(s) that is (are) involved in this pathway remain to be identified. Moreover, this pathway does not require RecA* for the proteolytic processing of a protein other than LexA (like the cleavage of UmuD in UV-induced mutagenesis). An "additional" role of RecA can be defined as follows: (i) The non-activated form of the RecA protein acts as an inhibitor in the NarI mutation pathway. (ii) This inhibition is relieved upon activation of RecA by UV irradiation of the bacteria. (iii) A recA deletion mutant is totally proficient in the NarI mutation pathway provided the SOS system is derepressed [lexA (Def) allele]. Therefore, RecA does not actively participate in the fixation of the mutation. A molecular model for this "additional" role of RecA is proposed.

Single adduct mutagenesis: strong effect of the position of a single acetylaminofluorene adduct within a mutation hot spot.

“…The mutation cross section at a Nar I site, based on the assumption of equal modification of the guanines (7), had been found to be -2.9 x 10-2 (6), a value somewhat higher than the currently determined 10-2. The mutation cross section at Nar I sites increases with the total extent of modification (8), suggesting the possible involvement of a two-hit mechanism when multiple adducts are present. The background mutation frequency introduced by the chemistry of the oligonucleotide and the engineering of the gapped-duplex construction is quite low.…”

“…Rather, the hot spot within the Nar I restriction site is the simplest member of a family of sequences that are specifically processed by a new mutation pathway. Characteristics of this pathway include the following: (i) it is under genetic control that differs from the pathway for base-pair substitution mutations (8) and from the Streisinger-type of frameshift mutations at repetitive sequences. An essential feature of the Nar I mutation pathway is its umuDC independence.…”

“…Preferential excision repair of the adducts at sites other than the hot spots can also be ruled out as an explanation for the existence of hot spots because the mutation spectra are similar in uvrA and wild-type strains (5). Mutagenesis at the Nar I sites has genetic requirements that differ from those of base-pair substitutions or frameshift mutations at repetitive sequences (5,8). Specifically, the so-called Nar I mutation pathway is UmuDC+ independent; although it requires the function of a lexA+-repressed gene, it does not require the activated form of RecA (RecA*) in its second role (8).…”

“…Mutagenesis at the Nar I sites has genetic requirements that differ from those of base-pair substitutions or frameshift mutations at repetitive sequences (5,8). Specifically, the so-called Nar I mutation pathway is UmuDC+ independent; although it requires the function of a lexA+-repressed gene, it does not require the activated form of RecA (RecA*) in its second role (8).…”

SOS factors involved in translesion synthesis

“…In contrast, we previously have described a frameshift mutation pathway that is SOS-inducible but umuDC independent (14)(15)(16). Frameshift mutations are efficiently induced by a large variety of chemical carcinogens that present an aromatic moiety.…”