DNA polymerase beta substrate specificity: side chain modulation of the "A-rule"

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Apurinic/apyrimidinic (AP) sites are common DNA lesions that can impede DNA synthesis and lead to mutations.
• DNA polymerases often insert deoxyadenosine monophosphate (dAMP) opposite AP sites, a process termed the A-rule.
• The role of specific amino acid residues in DNA polymerase active sites during AP site processing is not fully understood.

Purpose of the Study:

• To investigate the role of arginine residue 283 (Arg-283) in DNA polymerase beta (pol beta) in the preferential insertion of dAMP opposite AP sites.
• To elucidate the structural and mechanistic basis for dAMP insertion opposite abasic sites using crystallographic analysis.

Main Methods:

• Kinetic analysis of pol beta insertion opposite AP sites using wild-type and R283A mutant enzymes.
• X-ray crystallography to determine the structures of pol beta in complex with gapped DNA containing an AP site analog and a dATP analog.

Main Results:

• The R283A mutation in pol beta abolished the preferential incorporation of dAMP opposite an AP site.
• Crystallographic structures revealed that dATP analog binding induces a closed polymerase conformation and an unstable primer terminus.
• Structural data indicated an upstream shift of the templating residue, even without a template base, during dATP insertion opposite an abasic site.

Conclusions:

• Arginine 283 is critical for the A-rule in pol beta-mediated DNA repair.
• The insertion of dATP opposite abasic sites shares common mechanistic features with dATP misinsertions.
• Structural insights provide a molecular understanding of DNA polymerase fidelity at abasic sites.