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Effects of palindromes on in vivo DNA replication and mutagenesis in bacteriophage phi X174 RF DNA
U R Müller1, B K Perkins, W L Williams
1Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27858-4354.
Abstract:
Bacteriophage phi X174 mutants with insertions of palindromic DNA sequences are rapidly outgrown by competing wild-type phage (Müller & Turnage, J. Mol. Biol. 189: 285). The basis for this defect was investigated and found to be due to an exclusion event early in the infectious cycle, in which phage genomes with palindromic inserts were preferentially excluded by wt phage. In addition, we have obtained further evidence for a palindrome induced genetic instability. Both defects are dependent on palindrome size and sequence, consistent with a model which involves formation of cruciforms, or cruciform-like structures. We propose that formation of unusual DNA secondary structures reduces the effectiveness of replicative form (RF) DNA to interact with limiting replication factors or membrane binding sites, possibly because of interaction with the host recombination system.
Insights
Bacteriophage phi X174 mutants with palindromic DNA inserts are outcompeted by wild-type phage due to preferential exclusion and genetic instability. These defects stem from DNA secondary structures interfering with replication factors and host interactions.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Bacteriophage phi X174 mutants with inserted palindromic DNA sequences exhibit slower growth compared to wild-type phage.
- Previous research indicated competitive disadvantage for these mutants.
Purpose of the Study:
- To investigate the basis for the growth defect in bacteriophage phi X174 mutants with palindromic DNA inserts.
- To elucidate the mechanisms behind phage exclusion and genetic instability associated with palindromic sequences.
Main Methods:
- Comparative growth assays between mutant and wild-type phage.
- Analysis of phage-host interactions during the early infectious cycle.
- Investigation of DNA secondary structure formation (cruciforms) and its impact.
Main Results:
- Mutants with palindromic inserts are preferentially excluded by wild-type phage early in infection.
- Palindromic inserts induce genetic instability in the phage genome.
- Both exclusion and instability are dependent on the size and sequence of the palindrome.
Conclusions:
- Palindromic DNA inserts form cruciform structures, leading to phage exclusion and genetic instability.
- These DNA secondary structures likely interfere with replicative form (RF) DNA interactions with replication factors and membrane binding sites.
- Interaction with the host recombination system may contribute to the observed defects.