An in vitro assay for frameshift mutations: hotspots for deletions of 1 bp by Klenow-fragment polymerase share a consensus DNA sequence.

Abstract

The fidelity of in vitro DNA synthesis catalyzed by the large fragment of DNA polymerase I was examined. The templates, specifically designed to detect shifts to the +1 or to the -1 reading frame, are composites of M13mp8 and bacteriophage T4 rIIB DNA and were designed to assist in the identification of the types of frameshifts that are the specific consequence of DNA polymerization errors. In vitro polymerization by the Klenow fragment produced only deletions, rather than the mixture of duplications and deletions characteristic of in vivo frameshifts. The most frequent frameshifts were deletions of 1 bp opposite a template purine base. Hotspots for these deletions occurred when the template purine immediately preceded the template sequence TT. The highest mutation frequencies were seen when the TTPu consensus sequence was adjacent to G:C rich sequences in the 3' direction. The nature of the consensus sequence itself distinguishes this 1-bp deletion mechanism from those operating in DNA repeats and attributed to the misalignment of DNA primers during synthesis. Deletions that were larger than 1 or 2 bp isolated after in vitro replication were consistent with the misalignment of the primer. Deletions of 2 bp and complex frameshifts (the replacement of AA by C) were also found. Mechanisms that may account for these mutations are discussed.

Mechanism of Frameshift (Deletion) Generated by Acetylaminofluorene-Derived DNA Adducts in Vitro

“…So-called spontaneous frameshift deletions are formed during DNA replication, presumably induced by endogenous or exogenous DNA damage (reviewed in ref 1 ). The frequency of such deletions may be influenced by several factors, including DNA sequence context ( − ), the editing function of DNA polymerase ( , ), and imbalances in the dNTP pool ().…”

Misalignment-mediated DNA synthesis errors

“…This includes 221 different single-base substitutions at 114 different template positions (Kunkel & Alexander, 1986; , single-base frame shifts at 150 positions (Kunkel, 1986), and a variety of larger and/or more complex frame shifts. More recently, several assays have been designed to focus on particular subsets of frame-shift errors (de Boer & Ripley, 1988; Kunkel et al" 1989; Papanicolaou & Ripley, 1989; Bebenek . These assays use M13 DNA substrates that normally yield colorless plaques because they contain frame-shift mutations.…”

“…This possibility is supported by structural studies with oligonucleotides demonstrating that an extra base can exist in conformations that do not disrupt hydrogen bonding of adjacent base pairs [Patel et al, 1982;Hare et al, 1986;Roy et al, 1987;Woodson & Crothers, 1988;Joshua-Tor et al, 1988;Miller et al, 1988; for review, see Patel et al (1987)]. Extra bases may be stabilized by interactions with amino acids in the DNA polymerase (Kunkel, 1986; de Boer & Ripley, 1988;Miller et al, 1988), perhaps by formation of hydrogen bonds with specific nucleotides (Hendry et al, 1981(Hendry et al, , 1984Lacey & Mullins, 1983), which might exclude them from pairing with the other strand.…”

“…Models to explain the sequences of simple and complex frame-shift mutations recovered from reactions catalyzed by E. coli DNA polymerase I and its large fragment (de Boer & Ripley, 1988;Papanicolaou & Ripley, 1989) and yeast DNA polymerase I (Kunkel et al, 1989) also have invoked 1984), the ends of some complex deletions produced in vitro suggest the involvement of palindromic DNA sequence [again, see Figure 4 in Papanicolaou and Ripley (1989) or Figure 4 in Kunkel et al (1989)].…”

Ultrahigh-throughput screening-assisted in vivo directed evolution for enzyme engineering

“…On the one hand, the deletion included the primosome assembly signal (pas) site, probably resulting in more durable Pol I replication and mutation in sequences located further from ori [ 15 ]. On the other hand, it has been reported that both Pol I and mismatch repair-defective cells may lead to large indels, which probably results in this large fragment deletion [ 48 , 49 ]. In addition, the deletion mutation contains the gene encoding the Rop protein.…”