Replisome-mediated translesion synthesis by a cellular replicase.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Genome integrity is crucial and relies on the replisome's ability to handle DNA damage during replication.
• The Escherichia coli replisome can stall at DNA lesions, with options to restart replication downstream or use specialized polymerases for translesion synthesis.
• Previous studies hinted at a role for the E. coli replicase in lesion bypass, but this remained untested in reconstituted systems.

Purpose of the Study:

• To investigate whether the E. coli DNA polymerase III holoenzyme can directly bypass DNA lesions in a reconstituted replication system.
• To determine the efficiency and fidelity of lesion bypass by the replisome without specialized translesion synthesis polymerases.

Main Methods:

• Reconstituted DNA replication system using a stalled E. coli replisome.
• Introduction of specific DNA lesions (cyclobutane pyrimidine dimer, abasic site) into the template DNA.
• Analysis of replication bypass, efficiency, fidelity, and associated mutations.

Main Results:

• The DNA polymerase III holoenzyme directly bypassed cyclobutane pyrimidine dimers and abasic sites via translesion synthesis.
• Bypass efficiency correlated with deoxynucleotide concentrations and downstream primer synthesis frequency.
• Replication of cyclobutane pyrimidine dimers was accurate; abasic sites primarily caused -1 frameshifts.
• Lesion bypass increased base substitution frequency preceding the lesion and reduced lesion-skipping replication restart.

Conclusions:

• The E. coli replisome can directly perform translesion synthesis at DNA damage sites without recruiting specialized polymerases.
• This direct bypass mechanism is influenced by nucleotide availability and primer synthesis.
• While capable of bypass, the process can introduce replication errors, particularly frameshifts at abasic sites.