How a B family DNA polymerase has been evolved to copy RNA

Choi, Woo Suk; He, Peng; Pothukuchy, Arti; Gollihar, Jimmy; Ellington, Andrew D.; Yang, Wei

doi:10.1073/pnas.2009415117

Cited by 10 publications

(18 citation statements)

References 51 publications

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“…It is intriguing to observe how the DNA-dependent DNA polymerase and the RNA-dependent DNA polymerases are comparable structurally, although we cannot detect similarity in terms of primary sequence [ 23 ]. Another point to keep in mind is that some DNA-dependent DNA polymerases exhibit some reverse transcription activity and that only a few mutations can induce this activity to emerge in DNA-dependent DNA polymerases [ 29 , 30 , 31 ]. These facts, when taken together, may suggest that these two groups of proteins have a common ancestor.…”

Section: Resultsmentioning

confidence: 99%

“…In the process of replicating the genetic material, they act both in the replication of the genome and in the maturation of the Okazaki fragments, showing different functional versatility of the bacterial polymerases, since in this lineage, there was a specialization of the polymerases C and A for replication and maturation of Okazaki fragments, respectively. Some studies have shown that with a few mutation points, specimens of this family acquired a reverse transcriptase function, which may indicate an evolutionary reversion process, since it is suggested that all families of DNA-dependent DNA polymerases may have had a reverse transcriptase as an ancestral molecule [ 30 ]. In our analysis, sequences from the main groups of Archaea and Eukarya, and viral lineages, were used.…”

Section: Archaeal and Eukaryotic Dna-dependent Dna Polymerasementioning

confidence: 99%

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Natural History of DNA-Dependent DNA Polymerases: Multiple Pathways to the Origins of DNA

Farías

Furtado

Santos

et al. 2023

Viruses

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One of the major evolutionary transitions that led to DNA replacing RNA as the primary informational molecule in biological systems is still the subject of an intense debate in the scientific community. DNA polymerases are currently split into various families. Families A, B, and C are the most significant. In bacteria and some types of viruses, enzymes from families A and C predominate, whereas family B enzymes are more common in Archaea, Eukarya, and some types of viruses. A phylogenetic analysis of these three families of DNA polymerase was carried out. We assumed that reverse transcriptase was the ancestor of DNA polymerases. Our findings suggest that families A and C emerged and organized themselves when the earliest bacterial lineages had diverged, and that these earliest lineages had RNA genomes that were in transition—that is, the information was temporally stored in DNA molecules that were continuously being produced by reverse transcription. The origin of DNA and the apparatus for its replication in the mitochondrial ancestors may have occurred independently of DNA and the replication machinery of other bacterial lineages, according to these two alternate modes of genetic material replication. The family C enzymes emerged in a particular bacterial lineage before being passed to viral lineages, which must have functioned by disseminating this machinery to the other lineages of bacteria. Bacterial DNA viruses must have evolved at least twice independently, in addition to the requirement that DNA have arisen twice in bacterial lineages. We offer two possible scenarios based on what we know about bacterial DNA polymerases. One hypothesis contends that family A was initially produced and spread to the other lineages through viral lineages before being supplanted by the emergence of family C and acquisition at that position of the principal replicative polymerase. The evidence points to the independence of these events and suggests that the viral lineage’s acquisition of cellular replicative machinery was crucial for the establishment of a DNA genome in the other bacterial lineages, since these viral lineages may have served as a conduit for the machinery’s delivery to other bacterial lineages that diverged with the RNA genome. Our data suggest that family B initially established itself in viral lineages and was transferred to ancestral Archaea lineages before the group diversified; thus, the DNA genome must have emerged first in this cellular lineage. Our data point to multiple evolutionary steps in the origins of DNA polymerase, having started off at least twice in the bacterial lineage and once in the archaeal lineage. Given that viral lineages are implicated in a significant portion of the distribution of DNA replication equipment in both bacterial (families A and C) and Archaeal lineages (family A), our data point to a complex scenario.

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Section: Resultsmentioning

confidence: 99%

Section: Archaeal and Eukaryotic Dna-dependent Dna Polymerasementioning

confidence: 99%

Natural History of DNA-Dependent DNA Polymerases: Multiple Pathways to the Origins of DNA

Farías

Furtado

Santos

et al. 2023

Viruses

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“…KOD DNA polymerase (KOD pol) is an important representative of B family DNA polymerases 24,25 . Since the discovery and isolation of strain KOD1 by Morikawa et al in 1994 26 , an abundant number of related strains and their enzyme products were examined and reported from this archaeon 27,28 . Recombinantly produced KOD DNA polymerases are the key component of numerous biotechnological applications such as PCR 29 , RT-qPCR 30 , multiplex PCR 31 , incorporation of base/sugar modified nucleoside triphosphates 32 , and TNA synthesis 33,34,35 etc.…”

Section: Introductionmentioning

confidence: 99%

Semi-rational evolution of a recombinant DNA polymerase for modified nucleotide incorporation efficiency

Zhai

Wang²,

Liu³

et al. 2023

Preprint

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Engineering B family DNA polymerases to utilize artificial nucleotides with unique large group labeling is limited by a general lack of understanding about the mechanism of the structural determinants that define substrate specificity. In this study, we report the engineering process of a natural DNA polymerase (KOD pol) from Thermococcus Kodakaraenis by using semi-rational design strategies. The essential residues around the active pocket and DNA binding site of KOD pol are characterized by using an established high-throughput microwell-based screening method. We obtained a five-mutant variant by performing site-directed saturation mutagenesis and combinatorial mutations of motif A and position 485, including two previously reported mutation sites (141/143) with inactive exonuclease activity. Then we selected more mutants, which were expected to increase the catalytic activity based on computational simulations, to perform experimental verification. By stepwise combinatorial mutation, we obtained an eleven-mutation variant E10 with over 25 times improvement of kinetic efficiency which has similar and satisfactory performances on both the BGI NGS platform (SE50) and MGI CoolMPSTM platform (PE100). The results indicated that E10 is a potential candidate for commercialization. The beneficial mutation sites identified in this study might be shared with other archaeal B-family DNA polymerases, which can provide a guide for the mutagenesis of other types of enzymes.

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“…Because C-terminal ‘thumb’ domains of reverse transcriptase undergo significant conformational changes that are strictly required for polymerase activity ( 17 ), some studies have suggested that the telomerase CTE may also undergo changes from an open to a closed conformation during the catalytic cycle ( 18 ). For example, the study of the dynamics of TERT and TER during catalysis using single-molecule Förster resonance energy transfer (smFRET) ( 19 ), TERT modelling ( 14 ) and based on the structure of TERT in complexes with a BIBR1532 telomerase inhibitor ( 20 ) have suggested that CTE may undergo a conformational change.…”

Section: Introductionmentioning

confidence: 99%

Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi

Zhai

Réty

Chen

et al. 2021

Nucleic Acids Research

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Telomerase plays critical roles in cellular aging, in the emergence and/or development of cancer, and in the capacity for stem-cell renewal, consists of a catalytic telomerase reverse transcriptase (TERT) and a template-encoding RNA (TER). TERs from diverse organisms contain two conserved structural elements: the template-pseudoknot (T-PK) and a helical three-way junction (TWJ). Species-specific features of the structure and function of telomerase make obtaining a more in-depth understanding of the molecular mechanism of telomerase particularly important. Here, we report the first structural studies of N-terminally truncated TERTs from Candida albicans and Candida tropicalis in apo form and complexed with their respective TWJs in several conformations. We found that Candida TERT proteins perform only one round of telomere addition in the presence or absence of PK/TWJ and display standard reverse transcriptase activity. The C-terminal domain adopts at least two extreme conformations and undergoes conformational interconversion, which regulates the catalytic activity. Most importantly, we identified a conserved tertiary structural motif, called the U-motif, which interacts with the reverse transcriptase domain and is crucial for catalytic activity. Together these results shed new light on the structure and mechanics of fungal TERTs, which show common TERT characteristics, but also display species-specific features.

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How a B family DNA polymerase has been evolved to copy RNA

Cited by 10 publications

References 51 publications

Natural History of DNA-Dependent DNA Polymerases: Multiple Pathways to the Origins of DNA

Natural History of DNA-Dependent DNA Polymerases: Multiple Pathways to the Origins of DNA

Semi-rational evolution of a recombinant DNA polymerase for modified nucleotide incorporation efficiency

Crystal structures of N-terminally truncated telomerase reverse transcriptase from fungi

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