Related Experiment Videos
Replication inhibition and miscoding properties of DNA templates containing a site-specific cis-thymine glycol or
J M McNulty1, B Jerkovic, P H Bolton
1Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269, USA.
Abstract:
Oligodeoxynucleotides modified site-specifically with cis-thymine glycol or urea residue, two ionizing radiation/oxidation damages, were used as templates in primer extension reactions catalyzed by 3' --> 5' exonuclease-deficient Klenow fragment, human DNA polymerase beta, AMV reverse transcriptase, and a modified T7 DNA polymerase (Sequenase). Both lesions blocked DNA replication one nucleotide before and opposite the lesion site, but a significant fraction of full-length product was obtained after prolonged incubation. Hill plot analysis of the results on both thymine glycol- and urea- containing templates by 3' --> 5' exonuclease-deficient Klenow fragment for incorporation of either dATP or dGTP gave linear plots with Hill coefficients much less than 1. This suggests that the dNTP concentration influences the termination of DNA synthesis at multiple steps of the catalytic process. The specificity of nucleotide incorporation opposite these lesions and chain extension by the same polymerase was determined by a steady-state kinetic analysis. The kinetic studies established that the rate of nucleotide incorporation and chain extension was highest with deoxyadenosine opposite both these lesions. However, the efficiency of forming a G.T pair relative to an A.T pair for the control at a level of 1/10(9) was enhanced to approximately 1/160 for thymine glycol and 1/20 for urea, although the former lesion was more bypassable than the latter lesion. On the basis of these in vitro results, we conclude that both these DNA damages are impediments of DNA synthesis and that a urea residue, in particular, has the potential to miscode.
Insights
DNA damages like thymine glycol and urea block DNA replication. While deoxyadenosine incorporation is favored, urea residues may lead to miscoding during DNA synthesis.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Ionizing radiation and oxidation can cause DNA damage, including thymine glycol and urea residues.
- Understanding how DNA polymerases handle these lesions is crucial for DNA repair and replication fidelity.
Purpose of the Study:
- To investigate the impact of thymine glycol and urea lesions on DNA synthesis using various DNA polymerases.
- To determine the fidelity and efficiency of nucleotide incorporation opposite these DNA damages.
Main Methods:
- Primer extension assays using modified oligodeoxynucleotides containing site-specific thymine glycol or urea lesions.
- Enzymatic reactions catalyzed by exonuclease-deficient Klenow fragment, human DNA polymerase beta, AMV reverse transcriptase, and modified T7 DNA polymerase (Sequenase).
- Hill plot analysis and steady-state kinetic analysis to assess DNA synthesis termination and nucleotide incorporation fidelity.
Main Results:
- Both thymine glycol and urea lesions caused replication blockage one nucleotide before and opposite the lesion site.
- Prolonged incubation allowed for a significant fraction of full-length product synthesis.
- Kinetic studies revealed deoxyadenosine incorporation as the preferred nucleotide opposite both lesions.
- The efficiency of mispairing was higher for urea (1/20) compared to thymine glycol (1/160) relative to control A.T pairing.
Conclusions:
- Thymine glycol and urea residues act as impediments to DNA synthesis.
- Urea residues, in particular, demonstrate a potential for miscoding during DNA replication.
- DNA polymerase fidelity is influenced by the presence of oxidative DNA lesions.