Structure of the 2-Aminopurine-Cytosine Base Pair Formed in the Polymerase Active Site of the RB69 Y567A-DNA Polymerase

Reha-Krantz, Linda J.; Hariharan, Chithra; Subuddhi, Usharani; Xia, S.; Zhao, Chao; Beckman, Jeff; Christian, Thomas D.; Konigsberg, William H.

doi:10.1021/bi2014618

Cited by 29 publications

(28 citation statements)

References 59 publications

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“…The structure that we have reported for a dATP/dT-containing ternary complex of Y567A RB69pol shows that the minor groove HB network at the n-1 position of the template strand and the pol was disrupted by replacing Y567 with Ala. (37, 39) It is very likely that the Y567A RB69pol mutant was not able to sense the dA to 3DA substitution at this position. As predicted, the kinetic parameters for incorporation of dAMP opposite dT by the Y567A RB69pol mutant were almost identical for both control DNA and DNA with 3DA at the n-1 position of the template strand (Table 2).…”

Section: Results and Disscusionmentioning

confidence: 94%

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Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

et al. 2012

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Minor groove hydrogen bonding (HB) interactions between DNA polymerases and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer-extension. Since there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2’-deoxyadenosine (3DA), an analog of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady state kinetic parameters for the insertion of dAMP opposite dT using primer/template (P/T) containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 102–103 fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base-pair geometry of 3DA/dT is exactly the same as dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 102–103 fold decrease in nucleotide incorporation. The minor groove HB interactions between the n-2 position of the primer strand and RB69pol fixes the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands so that they are in the optinal orientation for DNA synthesis

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Section: Results and Disscusionmentioning

confidence: 94%

“…(39) The excitation and emission wavelengths for tC o are 364 nm and 450 nm respectively. The final reaction mixtures consisted of 50 mM Tris (pH7.4), 10 mM MgSO 4 , 200 nM P/T and 4 µM RB69pol.…”

Section: Methodsmentioning

confidence: 99%

Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

et al. 2012

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“…Human mitochondrial promoters present an extra challenge because the DNA region upstream of the transcription start-sites is GC rich, unlike the T7 and yeast mitochondrial promoters. Fortunately, the fluorescent base 2-AP pairs with both thymine and cytosine bases (Figure 4B ) ( 45 , 46 ) and hence can be used on human mitochondrial promoters. The desirable property of 2-AP is that its fluorescence is quenched in duplex DNA and enhanced when the 2-AP base pair is melted and unstacked.…”

Section: Resultsmentioning

confidence: 99%

Human mitochondrial transcription factors TFAM and TFB2M work synergistically in promoter melting during transcription initiation

et al. 2016

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Human mitochondrial DNA is transcribed by POLRMT with the help of two initiation factors, TFAM and TFB2M. The current model postulates that the role of TFAM is to recruit POLRMT and TFB2M to melt the promoter. However, we show that TFAM has ‘post-recruitment’ roles in promoter melting and RNA synthesis, which were revealed by studying the pre-initiation steps of promoter binding, bending and melting, and abortive RNA synthesis. Our 2-aminopurine mapping studies show that the LSP (Light Strand Promoter) is melted from −4 to +1 in the open complex with all three proteins and from −4 to +3 with addition of ATP. Our equilibrium binding studies show that POLRMT forms stable complexes with TFB2M or TFAM on LSP with low-nanomolar Kd values, but these two-component complexes lack the mechanism to efficiently melt the promoter. This indicates that POLRMT needs both TFB2M and TFAM to melt the promoter. Additionally, POLRMT+TFB2M makes 2-mer abortives on LSP, but longer RNAs are observed only with TFAM. These results are explained by TFAM playing a role in promoter melting and/or stabilization of the open complex on LSP. Based on our results, we propose a refined model of transcription initiation by the human mitochondrial transcription machinery.

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“…Our previous structural and kinetic studies with 2AP, a fluorescent adenine analog, have shown that 2AP fluorescence is quenched as dTTP binds to an RB69pol-P/T binary complex with 2AP as the templating base and a dideoxy-terminated ddC at the 3′-end of the primer (16,34). We have interpreted the fluorescence quenching by dTTP as a consequence of 2AP stacking with the penultimate base pair as the fingers domain closes.…”

Section: Resultsmentioning

confidence: 99%

Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics

Xia¹,

Wood²,

Bradley³

et al. 2013

Nucleic Acids Research

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Internal cavities are a common feature of many proteins, often having profound effects on the dynamics of their interactions with substrate and binding partners. RB69 DNA polymerase (pol) has a hydrophobic cavity right below the nucleotide binding pocket at the tip of highly conserved L415 side chain. Replacement of this residue with Gly or Met in other B family pols resulted in higher mutation rates. When similar substitutions for L415 were introduced into RB69pol, only L415A and L415G had dramatic effects on pre-steady-state kinetic parameters, reducing base selectivity by several hundred fold. On the other hand, the L415M variant behaved like the wild-type. Using a novel tCo-tCnitro Förster Resonance Energy Transfer (FRET) assay, we were able to show that the partition of the primer terminus between pol and exonuclease (exo) domains was compromised with the L415A and L415G mutants, but not with the L415M variant. These results could be rationalized by changes in their structures as determined by high resolution X-ray crystallography.

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Structure of the 2-Aminopurine-Cytosine Base Pair Formed in the Polymerase Active Site of the RB69 Y567A-DNA Polymerase

Cited by 29 publications

References 59 publications

Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

Human mitochondrial transcription factors TFAM and TFB2M work synergistically in promoter melting during transcription initiation

Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics

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