Loop II of DNA polymerase beta is important for polymerization activity and fidelity

The accurate replication and transmission of genetic information is critical in the life of an organism. During its entire lifespan, the genetic information is constantly under attack from endogenous and exogenous sources of damage. To ensure that the content of its genetic information is faithfully preserved for synthesis and transmission, eukaryotic cells have developed a complex system of genomic quality control. Key players in this process are DNA polymerases, the enzymes responsible for synthesizing the DNA, because errors introduced into the genome by polymerase can result in mutations. We use DNA polymerase beta (pol β) as a model system to investigate mechanisms of preserving fidelity during nucleotide incorporation. In the study described here, we characterized the role that loop II of pol β plays in maintaining the activity and fidelity of pol β. We report here that the absence or shortening of loop II compromises the catalytic activity of pol β. Our data also show that loop variants of a specific length have a lower fidelity when compared to the wild-type polymerase. Taken together, our results indicate that loop II is important for the catalytic activity and fidelity of pol β

The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient

Approximately 30% of human tumors characterized to date express DNA polymerase beta (pol β) variant proteins. Two of the polymerase beta cancer-associated variants are sequence-specific mutators, and one of them binds to DNA but has no polymerase activity. The Leu22Pro (L22P) DNA polymerase beta variant was identified in a gastric carcinoma. Leu22 resides within the 8 kDa amino terminal domain of DNA polymerase beta, which exhibits dRP lyase activity. This domain catalyzes the removal of deoxyribose phosphate during short patch base excision repair. We show that this cancer-associated variant has very little dRP lyase activity but retains its polymerase activity. Although residue 22 has no direct contact with the DNA, we report here that the L22P variant has reduced DNA-binding affinity. The L22P variant protein is deficient in base excision repair. Molecular dynamics calculations suggest that alteration of Leu22 to Pro changes the local packing, the loop connecting helices 1 and 2 and the overall juxtaposition of the helices within the N-terminal domain. This in turn affects the shape of the binding pocket that is required for efficient dRP lyase catalysis

Structural and mechanistic consequences of polypeptide binding by GroEL

The remarkable ability of the chaperonin GroEL to recognise a diverse range of non-native states of proteins constitutes one of the most fascinating molecular recognition events in protein chemistry. Recent structural studies have revealed a possible model for substrate binding by GroEL and a high-resolution image of the GroEL–GroES folding machinery has provided important new insights into our understanding of the mechanism of action of this chaperonin. Studies with a variety of model substrates reveal that the binding of substrate proteins to GroEL is not just a passive event, but can result in significant changes in the structure and stability of the bound polypeptide. The potential impact of this on the mechanism of chaperonin-assisted folding is not fully understood, but provides exciting scope for further experiment

Structural and Functional Characterisation of TesA - A Novel Lysophospholipase A from Pseudomonas aeruginosa

TesA from Pseudomonas aeruginosa belongs to the GDSL hydrolase family of serine esterases and lipases that possess a broad substrate- and regiospecificity. It shows high sequence homology to TAP, a multifunctional enzyme from Escherichia coli exhibiting thioesterase, lysophospholipase A, protease and arylesterase activities. Recently, we demonstrated high arylesterase activity for TesA, but only minor thioesterase and no protease activity. Here, we present a comparative analysis of TesA and TAP at the structural, biochemical and physiological levels. The crystal structure of TesA was determined at 1.9 Å and structural differences were identified, providing a possible explanation for the differences in substrate specificities. The comparison of TesA with other GDSL-hydrolase structures revealed that the flexibility of active-site loops significantly affects their substrate specificity. This assumption was tested using a rational approach: we have engineered the putative coenzyme A thioester binding site of E. coli TAP into TesA of P. aeruginosa by introducing mutations D17S and L162R. This TesA variant showed increased thioesterase activity comparable to that of TAP. TesA is the first lysophospholipase A described for the opportunistic human pathogen P. aeruginosa. The enzyme is localized in the periplasm and may exert important functions in the homeostasis of phospholipids or detoxification of lysophospholipids

Hydrophobic and Charged Residues in the C-Terminal Arm of Hepatitis C Virus RNA-Dependent RNA Polymerase Regulate Initiation and Elongation.

The RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a β-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of β-loop and C-terminal arm mutants, which were used for in vitro analysis of RdRp de novo initiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the β-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general. IMPORTANCE: HCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr→Phe or Asp→Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase

A membrane-bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli

Pseudomonas aeruginosa strain 1001 produces an esterase (EstA) that can hydrolyse the racemic methyl ester of b-acetylthioisobutyrate to produce the (D)-enantiomer, which serves as a precursor of captopril, a drug used for treatment of hypertension. We show here that PA2949 from P. aeruginosa PA01, a homologue of EstA, can efficiently be expressed in an enzymatically active form in E. coli. The enzyme is membrane-associated as demonstrated by cell fractionation studies. PA2949 was purified to homogeneity after solubilisation with the nonionic detergent, Triton X-100, and was shown to possess a conserved esterase catalytic triad consisting of Ser137–His258–Asp286. Our results should allow the development of an expression and purification strategy to produce this biotechnologically relevant esterase in a pure form with a high yield

Trafficability Prediction Using Depth-to-Water Maps : the Status of Application in Northern and Central European Forestry

DOI 10.1007/s40725-022-00159-w CorrectionPurpose of Review Mechanized logging operations with ground-based equipment commonly represent European production forestry but are well-known to potentially cause soil impacts through various forms of soil disturbances, especially on wet soils with low bearing capacity. In times of changing climate, with shorter periods of frozen soils, heavy rain fall events in spring and autumn and frequent needs for salvage logging, forestry stakeholders face increasingly unfavourable conditions to conduct low-impact operations. Thus, more than ever, planning tools such as trafficability maps are required to ensure efficient forest operations at reduced environmental impact. This paper aims to describe the status quo of existence and implementation of such tools applied in forest operations across Europe. In addition, focus is given to the availability and accessibility of data relevant for such predictions. Recent Findings A commonly identified method to support the planning and execution of machine-based operations is given by the prediction of areas with low bearing capacity due to wet soil conditions. Both the topographic wetness index (TWI) and the depth-to-water algorithm (DTW) are used to identify wet areas and to produce trafficability maps, based on spatial information. Summary The required input data is commonly available among governmental institutions and in some countries already further processed to have topography-derived trafficability maps and respective enabling technologies at hand. Particularly the Nordic countries are ahead within this process and currently pave the way to further transfer static trafficability maps into dynamic ones, including additional site-specific information received from detailed forest inventories. Yet, it is hoped that a broader adoption of these information by forest managers throughout Europe will take place to enhance sustainable forest operations.Peer reviewe

Trafficability Prediction Using Depth-to-Water Maps : the Status of Application in Northern and Central European Forestry

DOI 10.1007/s40725-022-00159-w CorrectionPurpose of Review Mechanized logging operations with ground-based equipment commonly represent European production forestry but are well-known to potentially cause soil impacts through various forms of soil disturbances, especially on wet soils with low bearing capacity. In times of changing climate, with shorter periods of frozen soils, heavy rain fall events in spring and autumn and frequent needs for salvage logging, forestry stakeholders face increasingly unfavourable conditions to conduct low-impact operations. Thus, more than ever, planning tools such as trafficability maps are required to ensure efficient forest operations at reduced environmental impact. This paper aims to describe the status quo of existence and implementation of such tools applied in forest operations across Europe. In addition, focus is given to the availability and accessibility of data relevant for such predictions. Recent Findings A commonly identified method to support the planning and execution of machine-based operations is given by the prediction of areas with low bearing capacity due to wet soil conditions. Both the topographic wetness index (TWI) and the depth-to-water algorithm (DTW) are used to identify wet areas and to produce trafficability maps, based on spatial information. Summary The required input data is commonly available among governmental institutions and in some countries already further processed to have topography-derived trafficability maps and respective enabling technologies at hand. Particularly the Nordic countries are ahead within this process and currently pave the way to further transfer static trafficability maps into dynamic ones, including additional site-specific information received from detailed forest inventories. Yet, it is hoped that a broader adoption of these information by forest managers throughout Europe will take place to enhance sustainable forest operations.Peer reviewe