A bipartite interaction with the processivity clamp potentiates Pol IV-mediated TLS.

Area of Science:

• Molecular Biology
• DNA Replication and Repair
• Biochemistry

Background:

• Processivity clamps, like the β2 clamp in E. coli, are essential for DNA replication and mediate polymerase switching during translesion synthesis (TLS).
• All three E. coli TLS polymerases utilize a conserved clamp-binding motif (CBM) for interaction with the β2 clamp, which is critical for TLS.
• E. coli Pol IV exhibits a unique secondary interaction, termed
• with the β2 clamp via non-CBM residues, whose functional significance is largely unknown.

Purpose of the Study:

• To investigate the functional role of the secondary "rim contact" between Pol IV and the β2 processivity clamp in translesion synthesis.
• To determine the importance of this rim contact for Pol IV-mediated TLS across different types of DNA replication blocks.

Main Methods:

• In vitro reconstitution assays to assess Pol IV-mediated TLS efficiency past defined DNA lesions (3-methyl dA and N2-furfuryl dG).
• Genetic analysis in E. coli cells harboring single copies of these DNA lesions to evaluate in vivo TLS performance.
• Analysis of replication fork dynamics and polymerase competition at lesion sites.

Main Results:

• Ablation of the Pol IV rim contact significantly impaired TLS past the strong replication block 3-methyl dA, while minimally affecting TLS past the weak block N2-furfuryl dG.
• Similar defects in handling strong replication blocks were observed in vivo in E. coli cells lacking the Pol IV rim interaction.
• The rim interaction, in conjunction with ssDNA binding protein, enhances Pol IV's ability to compete with Pol III for access to the β2 clamp's CBM at stalled replication forks.

Conclusions:

• The secondary rim contact is critical for Pol IV's efficient translesion synthesis activity specifically at challenging DNA replication blocks.
• This bipartite clamp interaction mechanism allows Pol IV to effectively displace Pol III and resolve stalled replication forks via TLS.
• The cooperative action of the rim contact and CBM interaction facilitates rapid TLS at the replication fork, thereby minimizing the generation of damage-induced mutations.