Barry W. Glickman

Missense mutation in the lacI gene of Escherichia coli: Inferences on the structure of the repressor protein

The lac repressor has been studied extensively but a precise three-dimensional structure remains unknown. Studies using mutational data can complement other information and provide insight into protein structure. We have been using the lacI gene-repressor protein system to study the mutational specificity of spontaneous and induced mutation. The sequencing of over 6000 lacI- mutations has revealed 193 missense mutations generating 189 amino acid replacements at 102 different sites within the lac repressor. Replacement sites are not distributed evenly throughout the protein, but are clustered in defined regions. Almost 40% of all sites and over one-half of all substitutions found occur within the amino-terminal 59 amino acid residues, which constitute the DNA-binding domain. The core domain (residues 60 to 360) is less sensitive to amino acid replacement. Here, substitution is found in regions involved in subunit aggregation and at sites surrounding residues that are implicated in sugar-binding. The distribution and nature of missense mutational sites directs attention to particular amino acid residues and residue stretches.

Mechanisms of spontaneous mutation in DNA repair-proficient Escherichia coli.

This paper describes the DNA sequence analysis of 729 independent spontaneous lacI- mutations. This total is comprised of 478 novel mutations and 251 previously described events, and therefore should allow a more comprehensive view of spontaneous mutation in Escherichia coli. The spectrum is dominated by a hotspot (71% of all events). Mutations at this site consist of related addition and deletion events involving a number of repetitive sequences. Here we discuss how the frequency and proportion of these events vary in different DNA repair-deficient genetic backgrounds. The distribution of non-hotspot events includes base substitutions (38%), deletions (35%), frameshifts (14%), duplications (4%) and insertion elements (4%). G:C----A:T events dominate among base substitutions, while G:C----C:G events are the least common; the remaining types of base substitution are equally represented. Among deletions, a significant number do not display repeated sequences at their endpoints (26/72). However, almost all multiply recovered events (15/17) possess repeated sequences capable of accounting for the deletion endpoints. Similarly, over half of all duplications recovered (5/7) display repeated endpoints. Single-base frameshifts are equally divided between A:T and G:C sites, in each case (-) 1 events occur 3-fold more frequently that (+) 1 events. A comparative analysis of each mutational class recovered to lacI- spectra available in a variety of DNA repair/metabolism-deficient strains is presented here in an attempt to assess possible contributions from chemical, physical and enzymic sources of damage.

Asymmetric cytosine deamination revealed by spontaneous mutational specificity in an Ung- strain of Escherichia coli.

SummaryA collection of 164 spontaneous lacI− mutations were recovered from a uracil-DNA glycosylase deficient (Ung−) strain of Escherichia coli and analyzed by DNA sequencing. As predicted by genetic studies, G:C→A:T transitions predominated among base substitution events. However, DNA sequence analysis indicated that these events did not occur at random. Of the 31 G:C→A:T transitions recovered, 24 involved cytosine residues located in the nontranscribed strand of the gene and 15 of the 31 transitions occurred at cytosines located on the 3′ side of 3 or more A:T base pairs. These differentials likely reflect the more single-stranded character of the non-transcribed strand of the gene and of regions rich in A:T base pairs. In addition, mutation at the frameshift hotspot was altered in the Ung− strain, suggesting a role for DNA repair in the formation of structural intermediates that potentiate these events. Also, the analysis of non-hotspot frameshifts, deletions and duplication showed that many involved local DNA sequence. Specifically, several of the frameshift, deletion and duplication mutations occurred near the sequence 5′-CTGG-3′. Thus, DNA sequence analysis of mutational specificity in an Ung− strain has provided evidence that gene expression, DNA repair and DNA context can all potentially influence the classes and frequencies of spontaneous mutation.

DNA sequence analysis of spontaneous mutation in a PolA1 strain of Escherichia coli indicates sequence-specific effects

SummaryThe sequences of a collection of 261 spontaneous lacI- mutants recovered in a PolA- strain of Escherichia coli have indicated an increase in the frequency of most classes of mutation in this strain. Among base substitutions in lacI, a preference for transversions over transitions was observed. In addition, a single transition in the lac operator was enhanced 8-fold. More significantly, of 18 frameshifts, 12 occurred adjacent to a 5′-GTGG-3′ sequence. Likewise, 15 of 24 deletions and 2 of 10 duplications had 5′-GTGG-3′ sequences at one or both endpoints. We speculate that the prevalence of mutations at these specific sequences reflects the persistence of strand discontinuities that enhance the opportunity for mutagenic mishaps. Further, 5′-GTGG-3′ sequences apparently represent sites where DNA polymerase I is involved in some aspect of DNA metabolism. These results strengthen the view that DNA context contributes an important component to spontaneous mutagenesis and indicate an anti-mutagenic role for DNA polymerase I.

Mutational analysis of the structure and function of the adenine phosphoribosyltransferase enzyme of Chinese hamster

We have analyzed the adenine phosphoribosyltransferase (APRT) enzyme from Chinese hamster ovary cells through the study of mutants that are able to grow in the presence of the toxic adenine analogue 8-azaadenine. The distribution of the amino acid alterations was analyzed in terms of the binding regions for the purine and phosphoribosylpyrophosphate substrates and a comparison was made with mutants known in human APRT and human, mouse and hamster hypoxanthine-guanine phosphoribosyltransferase. A number of mutants were found to cluster in several regions of the amino acid sequence. Residual enzyme activity with adenine was determined and this was correlated with substrate binding regions. A model of the secondary structure features is proposed.

Perspectives on UV light mutagenesis: investigation of the CHO aprt gene carried on a retroviral shuttle vector

The extent to which the cellular processing of shuttle vector-carried genes reflects that of endogenous chromosomal loci has been a subject of considerable controversy. In order to address this issue, we have developed a retroviral-based shuttle vector carrying the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase (aprt) gene stably integrated into the genome to be used for studying mutational specificity in mammalian cells. Initially, we have characterized a collection of UV-induced mutants in a CHO cell background. We have therefore been able to directly compare this shuttle vector data to that previously obtained for UV-induced mutation at the endogenous CHO (aprt)locus. Although some potential differences between the two spectra have been noted, there appears to be a remarkable similarity in the distribution and site specificity of UV-induced mutations. These similarities extend to extrachromosomal shuttle vectors as well and consolidate the role of shuttle vectors as powerful analytical tools for studying mechanisms of point mutagenesis in mammalian cells.

Differential enhancement of spontaneous transition mutations in the lacI gene of an Ung− strain of Escherichia coli

In this communication, the contribution of cytosine deamination to spontaneous mutagenesis in the lacI gene of E. coli was examined. In a wild-type strain, 75% of the amber mutations recovered were G:C----A:T transitions and 60% of these were at the 5-methylcytosine spontaneous hotspots Am6, Am15 and Am34. In a strain deficient for uracil-DNA glycosylase (Ung-), 96% of the amber mutations were G:C----A:T transitions while only 15% of these occurred at the hotspot sites. This shift in the mutational distribution demonstrates that cytosine deamination is a potent mutagenic process, which is enhanced in the absence of glycosylase. Moreover, some amber sites were greatly enhanced in the Ung- strain while others were only slightly enhanced. This result suggests that the rate of cytosine deamination at individual sites may be influenced by surrounding base composition. Therefore, we examined the neighboring sequences and found a strong correlation between the fold-increase in mutation and the A/T richness of the surrounding sequence. It is suggested that A/T-rich regions denature more often, forming transient single strands in which cytosine residues would be expected to deaminate more readily.

Mutagenesis by 8-methoxypsoralen plus near-UV treatment: analysis of specificity in the lacI gene of Escherichia coli.

We have studied the specificity of mutation induced by PUVA treatment in the lacI gene of E. coli. Cells were exposed to near UV (approximately 365 nm) in the presence of 8-methoxypsoralen under conditions yielding about 7% survival and a 10-fold increase in mutation frequency. The cloning and sequencing of 131 mutants recovered following PUVA treatment revealed that almost all classes of mutation including base substitutions, frameshifts and deletions were induced. The distribution of mutations was non-random and a region of the lacI gene was found to be virtually silent for all classes of mutation. Intriguingly, the broad spectrum of mutation is accompanied by the recovery of mutation at two spontaneous hotspots. We observed a 7-fold increase at a frameshift hotspot involving the gain or loss of a tetramer tandemly repeated 3 times at this site and a 23-fold increase at an A:T----G:C transition hotspot located at the +6 mutational spectrum recovered following PUVA treatment was unique and a detailed analysis of the different classes of mutations indicates a role for DNA repair of both monoadducts and cross-links in the production of mutation.

Specificity of recA441-mediated (tif-1) mutational events

SummaryTo investigate the impact of SOS induction on the distribution of spontaneous mutation, 111 recA441-mediated mutations were characterized at the DNA sequence level in the lacI gene of Escherichia coli. A 2.6-fold enhancement in lacI− mutation frequency was observed after induction of the SOS system in the absence of mutagenic treatment, and specific classes of mutational events were induced. G : C → C : G, G : C → T : A and A : T → T : A transversion events were specifically enhanced after SOS induction. A preferential 5′-Y-Purine-3′ neighbouring base specificity for these transversion events is reported here (normalised for mutation of the purine residue). In addition, a preference for transversion events at 5′-C/GTGG-3′ sequences is also observed. Fifty events were recovered at the lacI “frameshift hotspot site” and were equally represented by 4 bp addition and deletion events. This 1:1 ratio deviates significantly from the 4:1 distribution characteristic of spontaneous frameshift mutation in the RecA+ background and is a consequence of the fourfold induction of the (−)4 event. This abberrant distribution was confirmed by oligomeric probing of 474 independent recA441-mediated spontaneous lacI− mutations.

Mechanisms of Spontaneous Mutagenesis: Clues from Altered Mutational Specificity in DNA Repair-Defective Strains

Spontaneous mutation may be described as the net result of all that can go wrong with DNA during the life cycle of an organism. The student of mutation, however, views the world of mutation through a myriad of filters and sees only a fraction of the molecular events surrounding mutation. To begin with, the student sees only those changes that produce a selectable and hence observable alteration in phenotype. Moreover, the majority of errors made during DNA replication and the errors produced by the accumulation of DNA damage are corrected by the plethora of repair mechanisms that have evolved to maintain the accurate transmission of genetic material. Hence, the study of mutagenesis in strains defective in DNA repair can be expected to yield information about the sources of spontaneous mutation, both with respect to the errors avoided as well as to those errors made during attempts at repair. Our increasing knowledge about mechanisms of DNA repair and their influence on mutation, coupled with the newly developed ability to clone and sequence DNA containing mutations, provides an opportunity to explore the sources of spontaneous mutation.