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A bipartite interaction with the processivity clamp potentiates Pol IV-mediated TLS
Seungwoo Chang1, Luisa Laureti2, Elizabeth S Thrall1
1Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115.
Summary
The rim contact of DNA polymerase IV (Pol IV) with the β2 processivity clamp is crucial for translesion synthesis (TLS) past difficult DNA blocks, aiding rapid repair and reducing mutations.
Area of Science:
- Molecular Biology
- DNA Replication and Repair
- Biochemistry
Background:
- Processivity clamps, like the β2 clamp in Escherichia coli, are essential for DNA polymerases, particularly during translesion synthesis (TLS).
- TLS polymerases, including Pol IV, interact with the β2 clamp via a conserved clamp-binding motif (CBM).
- Pol IV also exhibits a weaker interaction with the clamp's rim, mediated by non-CBM residues, the functional significance of which was unclear.
Purpose of the Study:
- To investigate the role of the non-CBM rim contact between Pol IV and the β2 processivity clamp in TLS.
- To determine how this rim interaction contributes to resolving stalled replication forks at different types of DNA lesions.
Main Methods:
- In vitro reconstitution of the E. coli replisome to study TLS kinetics.
- Site-directed mutagenesis to ablate the Pol IV rim contact.
- Analysis of TLS efficiency past strong (3-deaza-methyl dA) and weak (N2-furfuryl dG) DNA lesions.
- In vivo studies using E. coli cells with single-copy genomic lesions.
Main Results:
- Ablation of the Pol IV rim contact significantly impaired TLS past a strong replication block (3-deaza-methyl dA) but had minimal effect on TLS past a weak block (N2-furfuryl dG).
- In vitro and in vivo experiments confirmed the critical role of the rim interaction for efficient TLS at challenging lesions.
- Single-stranded DNA binding protein facilitates Pol IV's low-affinity rim binding at stalled forks, enhancing its ability to compete with the replicative Pol III.
Conclusions:
- The bipartite interaction of Pol IV with the β2 clamp (CBM and rim contact) is crucial for efficient TLS, particularly at strong DNA blocks.
- The rim contact allows Pol IV to effectively compete with Pol III, ensuring rapid resolution of stalled replication forks.
- This mechanism enhances Pol IV's ability to perform TLS at difficult lesions, thereby minimizing DNA damage-induced mutagenesis.
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