Biology subjects

Nelson, S. W.

Publications and source records attributed to Nelson, S. W..

4 recordsLinked to original sources

bioRxiv2025

Single-Molecule FRET Reveals Two T4 Phage MR Complex Exonuclease States Regulated by the Mre11 Dimer Interface

The Mre11/Rad50 (MR) complex serves as an initial responder to double-stranded DNA breaks and is conserved across all domains of life. Rad50 possesses ATPase activity that provides energy for the nuclease activities of Mre11. To elucidate the variation in exonuclease activity across the T4 phage MR complex population, we conducted smFRET studies utilizing a dual-labeled Cy3 and Cy5 double-stranded DNA substrate. Our findings revealed that the wild-type (WT) MR complex population was predominantly divided into two distinct rates of exonuclease activity. The majority of the population (94%) exhibited rapid exonuclease activity, with an average rate exceeding 20 nucleotides per second, while the remaining fraction showed substantially slower exonuclease activity, averaging 0.50 nucleotides per second. Furthermore, we compared the activity of the WT population to a mutant complex harboring the Mre11 mutation L101D, which has previously been shown to disrupt the Mre11 dimer interface. The MR complex with the L101D mutation exhibited a much higher percentage of the population displaying slow exonuclease activity (78%), while the remaining population exhibited fast exonuclease activity at a rate similar to that of the WT population. Additional activity parameters were evaluated, such as pause times and transition amplitudes, under various enzyme concentrations, revealing further differences between the WT and L101D MR complexes. Our results provide new insights into the molecular mechanisms of MR complex exonuclease activity and suggest that the complex may adopt different conformations with distinct kinetic properties.

biochemistry↗

bioRxiv2022

Trypanosoma brucei Mitochondrial DNA Polymerase POLIB Contains a Novel Thumb Insertion That Confers Dominant Exonuclease Activity

Trypanosoma brucei and related parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of maxicircles and minicircles. The kDNA structure and replication mechanism are divergent and essential for parasite survival. POLIB is one of three Family A DNA polymerases independently essential to maintain the kDNA network. However, the division of labor among the paralogs, particularly which might fulfill the role of a replicative, proofreading enzyme remains enigmatic. De novo modeling of POLIB revealed a structure divergent from all other Family A polymerases in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3-5 exonucleases. Here we demonstrate recombinant POLIB 3-5 exonuclease prefers DNA vs. RNA substrates and degrades single- and double-stranded DNA in a non-processive manner. The exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB is able to extend an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either primer. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborate the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. The unique POLIB thumb insertion influences the balance between polymerase and exonuclease activity and provides another example of exquisite diversity among DNA polymerases for specialized function.

biochemistry↗

bioRxiv2022

Promising Antimalarials targeting Apicoplast DNA Polymerase from Plasmodium falciparum

Malaria is caused by the parasite Plasmodium falciparum, which contains an essential non-photosynthetic plastid called the apicoplast. A single DNA polymerase, apPOL, is targeted to the apicoplast, where it replicates and repairs the genome. apPOL has no direct orthologs in mammals and is considered a promising drug target for the treatment and/or prevention of malaria. We previously reported screening the Malaria Box to identify MMV666123 as an inhibitor of apPOL. Herein we extend our studies and report structure-activity relationships for MMV666123 and identify key structural motifs necessary for inhibition. Although attempts to crystallize apPOL with the inhibitor were not fruitful, kinetic analysis and crystal structure determinations of WT and mutant apo-enzymes, facilitated model building and provided insights into the putative inhibitor binding site. Our results validate apPOL as an antimalarial target and provide an avenue for the design of high potency, specific inhibitors of apPOL and other A-family DNA polymerases.

biochemistry↗

bioRxiv2022

Discovery of small molecule inhibitors of Plasmodium falciparum apicoplast DNA polymerase

Malaria is caused by infection with protozoan parasites of the Plasmodium genus, which is part of the phylum Apicomplexa. Most organisms in this phylum contain a relic plastid called the apicoplast. The apicoplast genome is replicated by a single DNA polymerase (apPOL), which is an attractive target for anti-malarial drugs. We screened small-molecule libraries (206,504 compounds) using a fluorescence-based high-throughput DNA polymerase assay. Dose/response analysis and counter-screening identified 186 specific apPOL inhibitors. Toxicity screening against human HepaRG human cells removed 84 compounds and the remaining were subjected to parasite killing assays using chloroquine resistant P. falciparum parasites. Nine compounds were potent inhibitors of parasite growth and may serve as lead compounds in efforts to discover novel malaria drugs.

biochemistry↗