A Nucleotide-Analogue-Induced Gain of Function Corrects the Error-Prone Nature of Human DNA Polymerase iota

Ketkar, Amit; Zafar, Maroof K.; Banerjee, Surajit; Márquez, Víctor E.; Egli, Martin; Eoff, Robert L.

doi:10.1021/ja304176q

Cited by 8 publications

(5 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For our docking submissions, we focused on the Y-family DNA polymerases. Multiple crystal structures have been reported for hpol η 1–437 , hpol κ 19–526 , and hpol ι 26–446 . ,,,− Both apo and binary coordinate files were submitted to SwissDock for each enzyme. For this discussion, we consider only the apo form since the only observed difference was in the binding pocket near the primer–template DNA/active site interface (pocket A).…”

Section: Discussionmentioning

confidence: 99%

Leukotriene Biosynthesis Inhibitor MK886 Impedes DNA Polymerase Activity

Ketkar

Zafar

Maddukuri

et al. 2013

Chem. Res. Toxicol.

Self Cite

View full text Add to dashboard Cite

Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments, and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ~16,000 compounds identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC50 values, the exception being a six- to eight-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. Based on Michaelis-Menten kinetic analyses and DNA binding assays the mechanism of inhibition by MK886 appears to be mixed. In silico docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886.

show abstract

Section: Discussionmentioning

confidence: 99%

Leukotriene Biosynthesis Inhibitor MK886 Impedes DNA Polymerase Activity

Ketkar

Zafar

Maddukuri

et al. 2013

Chem. Res. Toxicol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…An siRNA duplex containing a ribo-N-MC modification in the guide strand was taken up by the intracellular RNAi machinery and showed near-wildtype activity and target mRNA degradation (Terrazas et al 2011). A crystal structure of a N-MC-dATP nucleotide bound at the active site of the model DNA polymerase Dpo4 superimposed nearly perfectly with an unmodified dATP bound at the same site, demonstrating selectivity of the polymerase for the North substrate conformation (Ketkar et al 2012a), and no protein contacts to the cyclopropane ring were observed in either Dpo4 or human DNA polymerase iota (Ketkar et al 2012b). Both N-MC and S-MC nucleotides have been shown to reproduce the preferences of the corresponding unmodified nucleotides for rotamers at the sugar-base glycosidic bond (torsion angle χ) and ring-adjacent C3 ′ -C4 ′ bond (γ) (Marquez et al 1997(Marquez et al , 2004.…”

mentioning

confidence: 99%

Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

White

Sumita

Márquez

et al. 2018

RNA

Self Cite

View full text Add to dashboard Cite

In common with other self-cleaving RNAs, the lead-dependent ribozyme (leadzyme) undergoes dynamic fluctuations to a chemically activated conformation. We explored the connection between conformational dynamics and self-cleavage function in the leadzyme using a combination of NMR spin-relaxation analysis of ribose groups and conformational restriction via chemical modification. The functional studies were performed with a -methanocarbacytidine modification that prevents fluctuations to C2'-endo conformations while maintaining an intact 2'-hydroxyl nucleophile. Spin-relaxation data demonstrate that the active-site Cyt-6 undergoes conformational exchange attributed to sampling of a minor C2'-endo state with an exchange lifetime on the order of microseconds to tens of microseconds. A conformationally restricted species in which the fluctuations to the minor species are interrupted shows a drastic decrease in self-cleavage activity. Taken together, these data indicate that dynamic sampling of a minor species at the active site of this ribozyme, and likely of related naturally occurring motifs, is strongly coupled to catalytic function. The combination of NMR dynamics analysis with functional probing via conformational restriction is a general methodology for dissecting dynamics-function relationships in RNA.

show abstract

“…Recently we showed that another Y-family member, human DNA polymerase iota (hpol ι) utilizes N-MC-dATP more efficiently than the unmodified nucleotide. 38 Again, there were no protein contacts near the cyclopropane ring with either N-MC-dATP or S-MC-dATP bound to the hopl ι active site. The primary effect of the bicyclo[3.1.0]hexane moiety was to alter the syn/anti equilibrium such that the adenine group of N-MC-dATP preferentially adopted the anti conformation.…”

Section: ■ Discussionmentioning

confidence: 96%

“…The perturbations associated with S-MC-dADP binding to Dpo4 result from interactions with the 3′-OH group of the nucleotide and the steric gate residue, which wedges the incoming S-MC-dADP into the primer terminus and positions the phosphates poorly for catalysis. Recently we showed that another Y-family member, human DNA polymerase iota (hpol ι) utilizes N-MC-dATP more efficiently than the unmodified nucleotide (38). Again, there were no protein contacts near the cyclopropane ring with either N-MC-dATP or S-MC-dATP bound to the hopl ι active site.…”

Section: Discussionmentioning

confidence: 99%

Differential Furanose Selection in the Active Sites of Archaeal DNA Polymerases Probed by Fixed-Conformation Nucleotide Analogues

et al. 2012

Self Cite

View full text Add to dashboard Cite

DNA polymerases select for the incorporation of deoxyribonucleotide triphosphates (dNTPs) using amino acid side-chains that act as a “steric-gate” to bar improper incorporation of rNTPs. An additional factor in the selection of nucleotide substrates resides in the preferred geometry for the furanose moiety of the incoming nucleotide triphosphate. We have probed the role of sugar geometry during nucleotide selection by model DNA polymerases from Sulfolobus solfataricus using fixed conformation nucleotide analogues. North-methanocarba-dATP (N-MC-dATP) locks the central ring into a RNA-type (C2′-exo, North) conformation near a C3′-endo pucker and South-methanocarba-dATP (S-MC-dATP) locks the central ring system into a (C3′-exo, South) conformation near a C2′-endo pucker. Dpo4 preferentially inserts N-MC-dATP and in the crystal structure of Dpo4 in complex with N-MC-dAMP, the nucleotide analogue superimposes almost perfectly with Dpo4 bound to unmodified dATP. Biochemical assays indicate that the S. solfataricus B-family DNA polymerase Dpo1 can insert and extend from both N-MC-dATP and S-MC-dATP. In this respect, Dpo1 is unexpectedly more tolerant of substrate conformation than Dpo4. The crystal structure of Dpo4 bound to S-MC-dADP shows that poor incorporation of the Southern pucker by the Y-family polymerase results from a hydrogen bond between the 3′-OH group of the nucleotide analogue and the OH group of the steric gate residue, Tyr12, shifting the S-MC-dADP molecule away from the dNTP binding pocket and distorting the base pair at the primer-template junction. These results provide insights into substrate specificity of DNA polymerases, as well as molecular mechanisms that act as a barrier against insertion of rNTPs.

show abstract

A Nucleotide-Analogue-Induced Gain of Function Corrects the Error-Prone Nature of Human DNA Polymerase iota

Cited by 8 publications

References 51 publications

Leukotriene Biosynthesis Inhibitor MK886 Impedes DNA Polymerase Activity

Leukotriene Biosynthesis Inhibitor MK886 Impedes DNA Polymerase Activity

Coupling between conformational dynamics and catalytic function at the active site of the lead-dependent ribozyme

Differential Furanose Selection in the Active Sites of Archaeal DNA Polymerases Probed by Fixed-Conformation Nucleotide Analogues

Contact Info

Product

Resources

About