Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Evaluating the contribution of base stacking during translesion DNA replication.

Edmunds Z Reineks1, Anthony J Berdis

  • 1Department of Pharmacology and the Comprehensive Cancer Center, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.

Biochemistry
|January 14, 2004
PubMed
Summary

Researchers investigated why DNA polymerases often insert adenine opposite abasic sites, known as the A-rule. They found that base-stacking, not hydrogen bonding, drives efficient insertion of modified nucleotides opposite DNA lesions.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

The Hidden Cost of Hemolyzed Blood Samples in the Emergency Department.

The journal of applied laboratory medicine·2021
Same author

Emergency Nurses' Knowledge, Attitudes, and Practices Related to Blood Sample Hemolysis Prevention: An Exploratory Descriptive Study.

Journal of emergency nursing·2021
Same author

A Comparison of Five SARS-CoV-2 Molecular Assays With Clinical Correlations.

American journal of clinical pathology·2020
Same author

Seeing Red: Blood Sample Hemolysis Is Associated with Prolonged Emergency Department Throughput.

The journal of applied laboratory medicine·2020
Same author

A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

PLoS biology·2019
Same author

Administration of a Nucleoside Analog Promotes Cancer Cell Death in a Telomerase-Dependent Manner.

Cell reports·2018

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Enzymology

Background:

  • DNA damage, specifically abasic sites, poses a challenge for DNA replication.
  • The 'A-rule' describes the preferential insertion of deoxyadenosine monophosphate (dAMP) opposite abasic sites, despite their nontemplating nature.

Purpose of the Study:

  • To elucidate the molecular mechanism behind the 'A-rule' and preferential nucleotide insertion opposite abasic sites.
  • To evaluate the role of base-stacking and desolvation in translesion DNA synthesis.

Main Methods:

  • Utilized a kinetic approach to assess polymerization efficiency during translesion DNA replication.
  • Employed bacteriophage T4 DNA polymerase and measured the insertion of modified nucleotides opposite an abasic site.
  • Conducted pre-steady-state kinetic measurements to determine kinetic parameters (kpol and Kd).

Related Experiment Videos

Main Results:

  • Increasing nucleobase size did not correlate with increased insertion efficiency opposite abasic sites.
  • A 5-nitroindolyl-2 ahydro-deoxyriboside triphosphate analogue showed ~1000-fold greater insertion efficiency than dAMP.
  • Kinetic parameters for the analogue (kpol = 126 s⁻¹, Kd = 18 μM) rivaled natural base pair formation, indicating hydrogen bonding is not essential.
  • Differences in insertion rates were observed between T4 DNA polymerase and the Klenow fragment.

Conclusions:

  • Base-stacking and/or desolvation capabilities of incoming nucleobases, rather than hydrogen bonding, are critical for efficient DNA polymerization opposite abasic sites.
  • A model involving pi-pi stacking interactions between the incoming nucleobase and polymerase active site amino acids explains enhanced catalytic efficiency.
  • Enzyme structural differences account for variations in nucleotide insertion rates opposite abasic sites.