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A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates


Translesion synthesis (TLS) by Y-family DNA polymerases is a chief mechanism of DNA damage tolerance1. Such TLS can be accurate or error-prone, as it is for bypass of a cyclobutane pyrimidine dimer by DNA polymerase η (XP-V or Rad30) or bypass of a (6-4) TT photoproduct by DNA polymerase V (UmuD′2C), respectively2,3. Although DinB is the only Y-family DNA polymerase conserved among all domains of life, the biological rationale for this striking conservation has remained enigmatic4. Here we report that the Escherichia coli dinB gene is required for resistance to some DNA-damaging agents that form adducts at the N2-position of deoxyguanosine (dG). We show that DinB (DNA polymerase IV) catalyses accurate TLS over one such N2-dG adduct (N2-furfuryl-dG), and that DinB and its mammalian orthologue, DNA polymerase κ, insert deoxycytidine (dC) opposite N2-furfuryl-dG with 10–15-fold greater catalytic proficiency than opposite undamaged dG. We also show that mutating a single amino acid, the ‘steric gate’ residue of DinB (Phe13 → Val) and that of its archaeal homologue Dbh (Phe12 → Ala), separates the abilities of these enzymes to perform TLS over N2-dG adducts from their abilities to replicate an undamaged template. We propose that DinB and its orthologues are specialized to catalyse relatively accurate TLS over some N2-dG adducts that are ubiquitous in nature, that lesion bypass occurs more efficiently than synthesis on undamaged DNA, and that this specificity may be achieved at least in part through a lesion-induced conformational change.

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Figure 3: Importance of DinB F13 residue in vivo.
Figure 1: Bypass of N 2 -furfuryl-dG.
Figure 2: A single mutation in DinB or its archaeal orthologue Dbh separates their TLS and DNA polymerase activities.


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We thank C. Joyce and A. DeLucia for Dbh and Dbh(F12A); E. Friedberg, J. Stirman and P. Fischhaber for Mus musculus pol κ; T. Nohmi for pYG768 and pYG782; N. Geacintov for benzo[a]pyrene-damaged substrate; A. Banerjee for assistance with Supplementary Fig. S5; and J. Tuttle and J. Bowers for advice on protein purification. This work was supported by the NIH (grants to G.C.W. and J.M.E.). Author Contributions D.F.J. performed the protein purification and lesion bypass assays, and proposed the involvement of the steric gate residue in TLS. V.G.G. discovered the sensitivity of a ΔdinB strain to NFZ and 4-NQO and performed the mutagenesis experiments. J.C.D. constructed and purified the N2-furfuryl-dG-containing oligonucleotide substrate.

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Correspondence to Graham C. Walker.

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Supplementary information

Supplementary Figure 1

Sensitivity of strains bearing a deletion of dinB to NFZ and 4-NQO. (PPT 1354 kb)

Supplementary Figure 2

SDS–PAGE of purified native DinB. (PPT 543 kb)

Supplementary Figure 3

Structures of lesions used in this study. (PPT 71 kb)

Supplementary Figure 4

Schematic of running start, standing start, and mismatch extension assays. (PPT 24 kb)

Supplementary Figure 5

A homology model of DinB, constructed in part with SWISS-Model, reveals an N2-dG lesion binding pocket and suggests a mechanism for rate enhancement on a damaged template. (PPT 1410 kb)

Supplementary Figure 6

SDS–PAGE of purified DinB(F13V). (PPT 373 kb)

Supplementary Figure 7

Products of running start primer extension reactions (30 min) with 1, 10, and 50 nM DinB and DinB(F13V) on undamaged and tetrahydrofuran bearing templates reveal that the F13V mutation does not render DinB able to bypass this lesion. (PPT 3828 kb)

Supplementary Figure 8

Dominant negativity of pdinB(F13V). (PPT 410 kb)

Supplementary Figure 9

MALDI-TOF mass spectrometry of the N2-furfuryl-dG adduct. (PPT 64 kb)

Supplementary Figure Legends

Text to accompany the above Supplementary Figures. (DOC 30 kb)

Supplementary Methods

This describes strain and plasmid construction, protein purification, and detailed oligonucleotide substrate synthesis methods. (DOC 25 kb)

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Jarosz, D., Godoy, V., Delaney, J. et al. A single amino acid governs enhanced activity of DinB DNA polymerases on damaged templates. Nature 439, 225–228 (2006).

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