Discrimination against the Cytosine Analog tC by Escherichia coli DNA Polymerase IV DinB

Walsh, Jason; Bouamaïed, Imenne; Brown, Tom; Wilhelmsson, L. Marcus; Beuning, Penny J.

doi:10.1016/j.jmb.2011.03.069

Cited by 14 publications

(16 citation statements)

References 70 publications

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“…Primer extension assays were carried out as described previously [46] using 32 P-labeled 13-mer primer annealed to 18-mer template. Reactions contained a final concentration of 10 nM DNA polymerase, 100 nM primer/template DNA, 1 mM dNTPs or rNTPs, 7.5 mM MgSO 4 , 30 mM Hepes (pH 7.5), 20 mM NaCl, 2 mM β-mercaptoethanol, 1% (w/v) bovine serum albumin, and 4% glycerol.…”

Section: Methodsmentioning

confidence: 99%

Steric gate residues of Y-family DNA polymerases DinB and pol kappa are crucial for dNTP-induced conformational change

2015

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Discrimination against ribonucleotides by DNA polymerases is critical to preserve DNA integrity. For many DNA polymerases, including those of the Y family, rNTP discrimination has been attributed to steric clashes between a residue near the active site, the steric gate, and the 2′-hydroxyl of the incoming rNTP. Here we used hydrogen/deuterium exchange (HDX) mass spectrometry (MS) to probe the effects of the steric gate in the Y-family DNA polymerases Escherichia coli DinB and human DNA pol κ. Formation of a ternary complex with a G:dCTP base pair in the active site resulted in slower hydrogen exchange relative to a ternary complex with G:rCTP in the active site. The protection from exchange was localized to regions both distal and proximal to the active site, suggesting that DinB and DNA pol κ adopt different conformations depending on the sugar of the incoming nucleotide. In contrast, when the respective steric gate residues were mutated to alanine, the differences in HDX between the dNTP- and rNTP-bound ternary complexes were attenuated such that for DinB(F13A) and pol κ(Y112A), ternary complexes with either G:dCTP or G:rCTP base pairs had similar HDX profiles. Furthermore, the HDX in these ternary complexes resembled that of the rCTP-bound state rather than the dCTP-bound state of the wild-type enzymes. Primer extension assays confirmed that DinB(F13A) and pol κ(Y112A) do not discriminate against rNTPs to the same extent as the wild-type enzymes. Our observations indicate that the steric gate is crucial for rNTP discrimination because of its role in specifically promoting a dNTP-induced conformational change and that rNTP discrimination occurs in a relatively closed state of the polymerases.

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Section: Methodsmentioning

confidence: 99%

Steric gate residues of Y-family DNA polymerases DinB and pol kappa are crucial for dNTP-induced conformational change

2015

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“…It has been further shown that DinB is capable of adding dGTP opposite the modified pyrimidine 1,3-diaza-2-oxophenothiazine (tC) specifically but is incapable of continuing TLS beyond the modified base [91]. This is intriguing since DinB binds slightly more strongly to DNA primer/template constructs that contain the tC analog than it binds to an undamaged DNA primer/template, which may indicate a specific inability to bypass major-groove modified bases in DNA [91].…”

Section: Translesion Synthesis In E Colimentioning

confidence: 99%

“…This is intriguing since DinB binds slightly more strongly to DNA primer/template constructs that contain the tC analog than it binds to an undamaged DNA primer/template, which may indicate a specific inability to bypass major-groove modified bases in DNA [91]. However, it was also found that DinB inserts tC opposite G in the DNA template and is capable of extending from the newly-incorporated tC, suggesting that DinB shows asymmetric discrimination against the modified DNA template and the incoming nucleotide [91]. …”

Section: Translesion Synthesis In E Colimentioning

confidence: 99%

Multiple Strategies for Translesion Synthesis in Bacteria

Ippoliti

DeLateur

Jones

et al. 2012

Cells

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Damage to DNA is common and can arise from numerous environmental and endogenous sources. In response to ubiquitous DNA damage, Y-family DNA polymerases are induced by the SOS response and are capable of bypassing DNA lesions. In Escherichia coli, these Y-family polymerases are DinB and UmuC, whose activities are modulated by their interaction with the polymerase manager protein UmuD. Many, but not all, bacteria utilize DinB and UmuC homologs. Recently, a C-family polymerase named ImuC, which is similar in primary structure to the replicative DNA polymerase DnaE, was found to be able to copy damaged DNA and either carry out or suppress mutagenesis. ImuC is often found with proteins ImuA and ImuB, the latter of which is similar to Y‑family polymerases, but seems to lack the catalytic residues necessary for polymerase activity. This imuAimuBimuC mutagenesis cassette represents a widespread alternative strategy for translesion synthesis and mutagenesis in bacteria. Bacterial Y‑family and ImuC DNA polymerases contribute to replication past DNA damage and the acquisition of antibiotic resistance.

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“…Kinetic parameters V max and K m were determined by assessing the percentage of primer extended at various time points in the experiment and were derived using GraphPad Prism 1 nonlinear regression analysis software [Segel, 1993]. DNA binding experiments were conducted by titrating 25 lM annealed DNA into a 60 lL solution of 1 lM DinB as previously described [Walsh et al, 2011a]. Table I shows the top 8% of the POOL predicted residues from the homology model of DinB.…”

Section: Experimental Methodsmentioning

confidence: 99%

Effects of non‐catalytic, distal amino acid residues on activity of E. coli DinB (DNA polymerase IV)

Walsh

Parasuram

Rajput

et al. 2012

Environ and Mol Mutagen

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DinB is one of two Y family polymerases in E. coli and is involved in copying damaged DNA. DinB is specialized to bypass deoxyguanosine adducts that occur at the N 2 position, with its cognate lesion being the furfuryl adduct. Active site residues have been identified that make contact with the substrate and carry out deoxynucleotide triphosphate (dNTP) addition to the growing DNA strand. In DNA polymerases, these include negatively charged aspartate and glutamate residues (D8, D103, and E104 in E. coli DNA polymerase IV DinB). These residues position the essential magnesium ions correctly to facilitate nucleophilic attack by the primer hydroxyl group on the a-phosphate group of the incoming dNTP. To study the contribution of DinB residues to lesion bypass, the computational methods THEMATICS and POOL were employed. These methods correctly predict the known active site residues, as well as other residues known to be important for activity. In addition, these methods predict other residues involved in substrate binding as well as more remote residues. DinB variants with mutations at the predicted positions were constructed and assayed for bypass of the N 2 -furfuryl-dG lesion. We find a wide range of effects of predicted residues, including some mutations that abolish damage bypass. Moreover, most of the DinB variants constructed are unable to carry out the extension step of lesion bypass. The use of computational prediction methods represents another tool that will lead to a more complete understanding of translesion DNA synthesis. Environ. Mol. Mutagen. 53:766-776, 2012. V V C 2012 Wiley Periodicals, Inc.

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Discrimination against the Cytosine Analog tC by Escherichia coli DNA Polymerase IV DinB

Cited by 14 publications

References 70 publications

Steric gate residues of Y-family DNA polymerases DinB and pol kappa are crucial for dNTP-induced conformational change

Steric gate residues of Y-family DNA polymerases DinB and pol kappa are crucial for dNTP-induced conformational change

Multiple Strategies for Translesion Synthesis in Bacteria

Effects of non‐catalytic, distal amino acid residues on activity of E. coli DinB (DNA polymerase IV)

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