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Influence of DNA sequence on the nature of mispairing during DNA synthesis
1Verna & Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030.
Abstract:
A series of synthetic oligonucleotide primers, annealed at various positions along the lacZ-alpha region of bacteriophage M13mp9 template, were elongated by purified DNA polymerases in the presence of only 3 of the 4 deoxynucleoside triphosphates to achieve misincorporation at a total of 49 different positions along the template. The newly synthesized strands (containing misincorporated bases) were isolated and sequenced to determine the identity of misincorporated deoxynucleoside monophosphates. The results indicate that the kind of mispairing that occurs during DNA synthesis is greatly influenced by the nucleotide sequence of the template. Transition-type base substitutions predominated overall, but at many template positions, transversion-type base substitutions occurred, most commonly via A.A mispairing. The results of parallel determinations made with Escherichia coli DNA polymerase I ("large fragment" form) and DNA polymerase of Maloney murine leukemia virus indicated that, overall, the identity of polymerase had only a small effect on the kind of misincorporation that occurred at different positions along the template. However, at certain template positions, the nature of mispairing during DNA synthesis was reproducibly affected by differing polymerase active-site environment.
Insights
DNA polymerase misincorporation is influenced by template sequence, with transitions predominating. While polymerase type has minor effects, specific template positions can reproducibly alter mispairing during DNA synthesis.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA polymerases are crucial for DNA replication and repair, ensuring accurate base pairing.
- Mispairing during DNA synthesis can lead to mutations, impacting genetic stability.
- Understanding factors influencing misincorporation is key to comprehending mutation mechanisms.
Purpose of the Study:
- To investigate how template nucleotide sequence affects misincorporation by DNA polymerases.
- To determine the influence of different DNA polymerases on misincorporation patterns.
- To identify specific template positions that reproducibly alter mispairing.
Main Methods:
- Synthetic oligonucleotide primers were elongated using purified DNA polymerases with limited deoxynucleoside triphosphates.
- Mispairing was induced at 49 positions along a bacteriophage M13mp9 template.
- Newly synthesized DNA strands were sequenced to identify misincorporated bases and determine mispairing types.
Main Results:
- Template nucleotide sequence significantly influences the type of mispairing during DNA synthesis.
- Transition-type base substitutions were predominant, but transversion-type substitutions also occurred.
- Escherichia coli DNA polymerase I and Maloney murine leukemia virus polymerase showed similar overall misincorporation patterns.
- Specific template positions reproducibly altered mispairing, indicating polymerase active-site influence.
Conclusions:
- DNA synthesis misincorporation is primarily dictated by the template sequence.
- While polymerase identity has a limited effect, active-site environments can influence mispairing at specific sites.
- This study elucidates the sequence-dependent nature of DNA polymerase fidelity and misincorporation.