DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts.
Bottom Line: Pol IV has a unique ability to coexist with Pol III on the same β clamp and to positively dissociate Pol III from β clamp in a concentration-dependent manner.Reconstituting the entire process of TLS in vitro using E. coli replication machinery and Pol IV, we observed that a replication fork stalled at (-)-trans-anti-benzo[a]pyrene-N(2)-dG lesion on the leading strand was efficiently and quickly recovered via two sequential switches from Pol III to Pol IV and back to Pol III.Our results suggest that TLS by Pol IV smoothes the way for the replication fork with minimal interruption.
Affiliation: Division of Integrated Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.Show MeSH
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Mentions: Leading-strand synthesis continues across the blocking lesion by Pol IV-mediated TLS. (A) Reconstitution of TLS in oriC replication system. Template pMOL7-BP(−) or pMOL7-control was incubated at 30°C with Pol III (1.7 nM) and other replicative proteins. Pol IV (10 nM) was added at 5 min after the start of the reaction. Replication products obtained at 20 min were purified, divided and analyzed by alkaline agarose gel electrophoresis (Figure 1), sequence gel electrophoresis (Figure 2) and 2D gel electrophoresis (Figure 4). Possible product structures are depicted in the panels on the right. (B) The 32P-labeled replication products of pMOL7-BP(−) (lanes 2, 4, 6 and 8) or pMOL7-control (lanes 3, 5, 7 and 9) in the absence (lanes 2, 3, 8 and 9) or presence of Pol IV (wild-type, in lanes 4 and 5; D8A-mutant, in lanes 6 and 7) were separated on an alkaline agarose gel. Size markers (M, 5′-32P-labeled λ/EcoT14I) are indicated in lane 1.
Affiliation: Division of Integrated Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.