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J Biol Chem. 2019 May 10;294(19):7588-7600. doi: 10.1074/jbc.RA118.006233. Epub 2019 Mar 14.

Residues in the fingers domain of the translesion DNA polymerase DinB enable its unique participation in error-prone double-strand break repair.

Author information

1
From the Department of Biology, Northeastern University, Boston, Massachusetts 02115.
2
the Department of Physics, Harvard University, Cambridge, Massachusetts 02138, and.
3
the Laboratoire de Biochimie Théorique, CNRS UPR9080 and Université Paris Diderot, IBPC, 75005 Paris, France.
4
From the Department of Biology, Northeastern University, Boston, Massachusetts 02115, v.godoycarter@northeastern.edu.

Abstract

The evolutionarily conserved Escherichia coli translesion DNA polymerase IV (DinB) is one of three enzymes that can bypass potentially deadly DNA lesions on the template strand during DNA replication. Remarkably, however, DinB is the only known translesion DNA polymerase active in RecA-mediated strand exchange during error-prone double-strand break repair. In this process, a single-stranded DNA (ssDNA)-RecA nucleoprotein filament invades homologous dsDNA, pairing the ssDNA with the complementary strand in the dsDNA. When exchange reaches the 3' end of the ssDNA, a DNA polymerase can add nucleotides onto the end, using one strand of dsDNA as a template and displacing the other. It is unknown what makes DinB uniquely capable of participating in this reaction. To explore this topic, we performed molecular modeling of DinB's interactions with the RecA filament during strand exchange, identifying key contacts made with residues in the DinB fingers domain. These residues are highly conserved in DinB, but not in other translesion DNA polymerases. Using a novel FRET-based assay, we found that DinB variants with mutations in these conserved residues are less effective at stabilizing RecA-mediated strand exchange than native DinB. Furthermore, these variants are specifically deficient in strand displacement in the absence of RecA filament. We propose that the amino acid patch of highly conserved residues in DinB-like proteins provides a mechanistic explanation for DinB's function in strand exchange and improves our understanding of recombination by providing evidence that RecA plays a role in facilitating DinB's activity during strand exchange.

KEYWORDS:

DNA damage; DNA polymerase; DNA polymerase IV; DNA repair; DNA synthesis; DinB; RecA; homologous recombination

PMID:
30872406
PMCID:
PMC6514613
[Available on 2020-05-10]
DOI:
10.1074/jbc.RA118.006233

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