Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Ultraviolet (UV) radiation induces DNA damage, primarily cyclobutane pyrimidine dimers (CPDs).
• CPDs are significant roadblocks to DNA replication, leading to cell death if unrepaired.
• Understanding the structural mechanisms of DNA polymerases interacting with CPDs is crucial for comprehending replication fidelity and bypass mechanisms.

Purpose of the Study:

• To elucidate the structural basis by which a replicative DNA polymerase encounters and processes CPDs during DNA replication.
• To provide atomic-level insights into the polymerase-DNA interactions at the site of UV-induced damage.

Main Methods:

• X-ray crystallography was employed to determine high-resolution structures.
• Structures were obtained for bacteriophage T7 DNA polymerase in complex with nucleotide substrates and a DNA template containing a cis-syn CPD.

Main Results:

• The DNA polymerase adopts different conformations depending on the position of the CPD within the active site.
• When the 3' thymine of the CPD is templating, the lesion is extruded, and the polymerase fingers subdomain opens.
• When the 5' thymine of the CPD is templating, the CPD is accommodated in the active site, base-pairing with the primer's 3' base and the incoming nucleotide, with closed fingers.

Conclusions:

• The determined structures reveal distinct mechanisms by which the T7 DNA polymerase interacts with CPDs.
• These structural insights explain the potent stalling of DNA replication caused by CPDs.
• The findings contribute to understanding DNA repair and replication bypass strategies at damaged DNA sites.