DNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Palanivelu, Peramachi

doi:10.9734/ijbcrr/2013/3777

Cited by 7 publications

(20 citation statements)

References 20 publications

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“…It has been found that almost all DNA polymerases and SSU RNAPs use an invariant K for catalysis, i.e., in the initial proton transfer reactions [34,29]. However, in all MSU RNAPs analyzed, no K was found at the expected distance from the template binding YG/FG pair but an equivalent invariant R (Table 4).…”

Section: Catalytic Regionmentioning

confidence: 98%

“…The absolutely conserved R, which is implicated in NTP selection in SSU and MSU RNAPs and DNA polymerases, is placed at -5/6 positions. In fact, in all the eubacterial β subunits the catalytic R is placed at -7 th position from the YG pair and completely conserved R is placed at -8 th position downstream from the catalytic R. However, catalytic R is placed at -8 th position from the YG pair the completely conserved R was at -4 th position in SSU RNAPs and DNA polymerases [29,34]. This strongly suggests that the DNA polymerases, SSU and MSU RNAPs use the same set of amino acids for template, substrate binding and catalysis establishing a structurefunction relationship among the DNA and RNA polymerases.…”

Section: Msa Of Eukaryotic Msu Rna Polymerases From Different Sourcesmentioning

confidence: 99%

“…This strongly suggests that the DNA polymerases, SSU and MSU RNAPs use the same set of amino acids for template, substrate binding and catalysis establishing a structurefunction relationship among the DNA and RNA polymerases. The immediate upstream amino acid from catalytic K in DNA polymerases is usually a G or A [34], but in SSU viral RNA polymerases it is a Q [29] and in MSU eubacterial β subunits, it is a D in all [4] and in eukaryotic Rpb2 it is S/T, suggesting a possible role in the substrate binding and catalysis. Another catalytic like region is located in ~400 amino acids from the N-terminal but with a YG/FG pair and a catalytic R at -9 and long highly conserved stretches on both the sides.…”

Section: Msa Of Eukaryotic Msu Rna Polymerases From Different Sourcesmentioning

confidence: 99%

“…A completely conserved R found upstream from the catalytic R is located at -6 th position which is implicated in NTP selection. It is interesting to note a completely conserved R found upstream from the catalytic R is missing in eubacteria (Table 1) [29,34]. The immediate downstream amino acid from catalytic K in DNA polymerases is usually a G or A [29], but in viral RNA polymerases it is a K or R, in MSU β' subunits, it is a D and in all β' subunits it is an S and it is an S/T in eukaryotes suggesting a possible role in NTP selection.…”

Section: Msa Of Eukaryotic Msu Rna Polymerases From Different Sourcesmentioning

confidence: 99%

“…However, in all MSU RNAPs analyzed, no K was found at the expected distance from the template binding YG/FG pair but an equivalent invariant R (Table 4). However, a detailed analysis has shown that all prokaryotic DNA polymerases II also use an invariant R in catalysis with similar distance conservations [34,4] instead of a usual K; interestingly, an enzyme also possesses primase activity and along with associated 3′→5′ exonuclease activity. Table 4 shows the invariant template binding YG pair with its catalytic R in the initiation (β and Rpb2) and in the elongation subunits (β' and Rpb1) of eubacterial and eukaryotic MSU RNAPs, respectively (Figs.…”

Section: Catalytic Regionmentioning

confidence: 99%

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Eukaryotic Multi-subunit DNA dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Palanivelu

2019

IJBcRR

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Aim: To analyze the most complex multi-subunit (MSU) DNA dependent RNA polymerases (RNAPs) of eukaryotic organisms and find out conserved motifs, metal binding sites and catalytic regions and propose a plausible mechanism of action for these complex eukaryotic MSU RNAPs, using yeast (Saccharomyces cerevisiae) RNAP II, as a model enzyme. Study Design: Bioinformatics, Biochemical, Site-directed mutagenesis and X-ray crystallographic data were analyzed. Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007- 2013. Methodology: Bioinformatics, Biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data of the enzyme were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of the MSU DNA dependent RNAPs from various eukaryotic sources. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical and SDM experiments and X-ray crystallographic analysis of these enzymes were used to confirm the possible amino acids involved in the active sites and catalysis. Results: Multiple sequence alignment (MSA) of RNAPs from different eukaryotic organisms showed a large number of highly conserved motifs among them. Possible catalytic regions in the catalytic subunits of the yeast Rpb2 (= β in eubacteria) and Rpb1 (= β’ in eubacteria) consist of an absolutely conserved amino acid R, in contrast to a K that was reported for DNA polymerases and single subunit (SSU) RNAPs. However, the invariant ‘gatekeeper/DNA template binding’ YG pair that was reported in all SSU RNAPs, prokaryotic MSU RNAPs and DNA polymerases is also highly conserved in eukaryotic Rpb2 initiation subunits, but unusually a KG pair is found in higher eukaryotes including the human RNAPs. Like the eubacterial initiation subunits of MSU RNAPs, the eukaryotic initiation subunits, viz. Rpb2, exhibit very similar active site and catalytic regions but slightly different distance conservations between the template binding YG/KG pair and the catalytic R. In the eukaryotic initiation subunits, the proposed catalytic R is placed at the -9th position from the YG/KG pair and an invariant R is placed at -5 which are implicated to play a role in nucleoside triphosphate (NTP) selection as reported for SSU RNAPs (viral family) and DNA polymerases. Similarly, the eukaryotic elongation subunits (Rpb1) are also found to be very much homologous to the elongation subunits (β’) of prokaryotes. Interestingly, the catalytic regions are highly conserved, and the metal binding sites are absolutely conserved as in prokaryotic MSU RNAPs. In eukaryotes, the template binding YG pair is replaced with an FG pair. Another interesting observation is, similar to the prokaryotic β’ subunits, in the eukaryotic Rpb1 elongation subunits also, the proposed catalytic R is placed double the distance, i.e., -18 amino acids downstream from the FG pair unlike in the SSU RNAPs and DNA polymerases where the distance is only -8 amino acids downstream from the YG pair. Thus, the completely conserved FG pair, catalytic R with an invariant R, at -6th position are proposed to play a crucial role in template binding, NTP selection and polymerization reactions in the elongation subunits of eukaryotic MSU RNAPs. Moreover, the Zn binding motif with the three completely conserved Cs is also highly conserved in the eukaryotic elongation subunits. Another important difference is that the catalytic region is placed very close to the N-terminal region in eukaryotes. Conclusions: Unlike reported for the DNA polymerases and SSU RNA polymerases, the of eukaryotic MSU RNAPs use an R as the catalytic amino acid and exhibit a different distance conservation in the initiation and elongation subunits. An invariant Zn2+ binding motif found in the Rpb1 elongation subunits is proposed to participate in proof-reading function. Differences in the active sites of bacterial and human RNA polymerases may pave the way for the design of new and effective drugs for many bacterial infections, including the multidrug resistant strains which are a global crisis at present.

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Section: Catalytic Regionmentioning

confidence: 98%

Section: Msa Of Eukaryotic Msu Rna Polymerases From Different Sourcesmentioning

confidence: 99%

Section: Msa Of Eukaryotic Msu Rna Polymerases From Different Sourcesmentioning

confidence: 99%

Section: Msa Of Eukaryotic Msu Rna Polymerases From Different Sourcesmentioning

confidence: 99%

Section: Catalytic Regionmentioning

confidence: 99%

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Eukaryotic Multi-subunit DNA dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Palanivelu

2019

IJBcRR

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The Arabidopsis DNA Polymerase δ Has a Role in the Deposition of Transcriptionally Active Epigenetic Marks, Development and Flowering

et al. 2015

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DNA replication is a key process in living organisms. DNA polymerase α (Polα) initiates strand synthesis, which is performed by Polε and Polδ in leading and lagging strands, respectively. Whereas loss of DNA polymerase activity is incompatible with life, viable mutants of Polα and Polε were isolated, allowing the identification of their functions beyond DNA replication. In contrast, no viable mutants in the Polδ polymerase-domain were reported in multicellular organisms. Here we identify such a mutant which is also thermosensitive. Mutant plants were unable to complete development at 28°C, looked normal at 18°C, but displayed increased expression of DNA replication-stress marker genes, homologous recombination and lysine 4 histone 3 trimethylation at the SEPALLATA3 (SEP3) locus at 24°C, which correlated with ectopic expression of SEP3. Surprisingly, high expression of SEP3 in vascular tissue promoted FLOWERING LOCUS T (FT) expression, forming a positive feedback loop with SEP3 and leading to early flowering and curly leaves phenotypes. These results strongly suggest that the DNA polymerase δ is required for the proper establishment of transcriptionally active epigenetic marks and that its failure might affect development by affecting the epigenetic control of master genes.

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Identification of Polymerase and Proofreading Exonuclease Domains in the DNA Polymerases IA, IB and Nuclear-Encoded RNA Polymerase of the Plant Chloroplasts

Palanivelu¹

2023

World J. Adv. Res. Rev.

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Chloroplast plays a crucial role in all photosynthetic plants and converts the light energy to chemical energy. It is a semi-autonomous organelle and is mostly controlled by its own genome and partly by the nuclear imports. To replicate its own genome, it uses two DNA polymerases, viz. polymerases IA and IB. DNA polymerase IA showed 72.45% identity to polymerase IB, but only 35.35% identity to the E. coli DNA polymerase I. Multiple sequence alignment (MSA) analysis have shown that the DNA polymerases IA and IB and the E. coli DNA polymerase I possess almost identical active sites for polymerization and proofreading (PR) functions, suggesting their possible common evolutionary origin. The nuclear-encoded RNA polymerase (NEP) is imported from the nucleus and involves in the transcription of all the four subunits of the chloroplast RNA polymerase. The polymerase catalytic core of the DNA polymerases IA, IB and the NEP are remarkably conserved and is in close agreement with other DNA/RNA polymerases reported already, and possess a typical template-binding pair (-YG-), a basic catalytic amino acid (K) to initiate catalysis and a basic nucleotide selection amino acid R at -4 from K. The DNA polymerases IA and IB are very similar to prokaryotic DNA polymerases, except in possessing a zinc-binding motif (ZBM) in them, like the eukaryotic replicases. Interestingly, the PR exonucleases of all three polymerases belong to the DEDD-superfamily of exonucleases. The DNA polymerases IA and IB belong to the DEDD(Y)-subfamily, whereas the NEP belongs to the DEDD(H)-subfamily.

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DNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Cited by 7 publications

References 20 publications

Eukaryotic Multi-subunit DNA dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

Eukaryotic Multi-subunit DNA dependent RNA Polymerases: An Insight into Their Active Sites and Catalytic Mechanism

The Arabidopsis DNA Polymerase δ Has a Role in the Deposition of Transcriptionally Active Epigenetic Marks, Development and Flowering

Identification of Polymerase and Proofreading Exonuclease Domains in the DNA Polymerases IA, IB and Nuclear-Encoded RNA Polymerase of the Plant Chloroplasts

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