Purification and characterization of novel X and Y-family DNA polymerases

DNA polymerases are known to be responsible for the replication and maintenance of an organism’s genomic DNA. However, with the completion of the human genome project, roughly ten additional DNA polymerases have been identified, and in many cases the physiological role of these novel enzymes remains to be elucidated. DNA polymerase lambda (Pol λ) is one such enzyme, and while it shares 54% sequence homology with the well-characterized DNA polymerase beta (Pol β), both genetic and biochemical evidence suggest a more complex physiological role for Pol λ. Pol λ demonstrates biochemical properties that are unprecedented in all characterized DNA polymerases with the most notable being the tight binding of all correct and incorrect canonical deoxynucleoside triphosphates (dNTP’s). DNA polymerases commonly demonstrate a preference for the “correct” incoming nucleotide by a significantly tighter binding of that nucleotide which results in a correct incorporation. Pol λ, however, binds all possible incoming nucleotides with a similar and tight ground-state binding affinity. By examining the crystal structure of truncated Pol λ several active site residues potentially involved in nucleotide binding were identified. Then, using site-directed mutagenesis, a series of mutant enzymes were generated to probe the structural basis of this phenomenon. Finally, pre-steady-state kinetics were employed to measure the ground state binding affinity for correct and incorrect incoming nucleotides in each of these mutant enzymes. Our results indicate that multiple amino acid residues are responsible for tight nucleotide binding in Pol λ. Residue R386 stabilizes the incoming nucleotide by salt bridge formation between the gamma phosphate of the nucleotide and the positively-charged guanidinium group on arginine, while residues A510 and R514 participate in van der Waal’s interactions with the base and sugar of the incoming nucleotide. Advisor: Zucai Su

Crystal Structures of Acyl Carrier Protein in Complex with Two Catalytic Partners Show a Dynamic Role in Cellular Metabolism

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/107352/1/1079_ftp.pd

Control of Stereoselectivity in Diverse Hapalindole Metabolites is Mediated by Cofactor‐Induced Combinatorial Pairing of Stig Cyclases

Stereospecific polycyclic core formation of hapalindoles and fischerindoles is controlled by Stig cyclases through a three‐step cascade involving Cope rearrangement, 6‐exo‐trig cyclization, and a final electrophilic aromatic substitution. Reported here is a comprehensive study of all currently annotated Stig cyclases, revealing that these proteins can assemble into heteromeric complexes, induced by Ca2+, to cooperatively control the stereochemistry of hapalindole natural products.Die stereospezifische Bildung des polycyclischen Kerns der Hapalindole und Fischerindole wird durch Stig‐Cyclasen gesteuert, die eine dreistufige Kaskade aus Cope‐Umlagerung, 6‐exo‐trig‐Cyclisierung und elektrophiler aromatischer Substitution vermitteln. Die Proteine können sich induziert durch Ca2+ zu heterotrimeren Komplexen zusammenlagern, um auf kooperative Weise die Stereochemie zu steuern.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/155506/1/ange201913686.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155506/2/ange201913686-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/155506/3/ange201913686_am.pd

Structural and stereochemical diversity in prenylated indole alkaloids containing the bicyclo[2.2.2]diazaoctane ring system from marine and terrestrial fungi

Various fungi of the generaAspergillus,Penicillium, andMalbrancheaproduce prenylated indole alkaloids possessing a bicyclo[2.2.2]diazaoctane ring system.</p

Mechanism of double-base lesion bypass catalyzed by a Y-family DNA polymerase

As a widely used anticancer drug, cis-diamminedichloroplatinum(II) (cisplatin) reacts with adjacent purine bases in DNA to form predominantly cis-[Pt(NH3)2{d(GpG)-N7(1),-N7(2)}] intrastrand cross-links. Drug resistance, one of the major limitations of cisplatin therapy, is partially due to the inherent ability of human Y-family DNA polymerases to perform translesion synthesis in the presence of DNA-distorting damage such as cisplatin–DNA adducts. To better understand the mechanistic basis of translesion synthesis contributing to cisplatin resistance, this study investigated the bypass of a single, site-specifically placed cisplatin-d(GpG) adduct by a model Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4). Dpo4 was able to bypass this double-base lesion, although, the incorporation efficiency of dCTP opposite the first and second cross-linked guanine bases was decreased by 72- and 860-fold, respectively. Moreover, the fidelity at the lesion decreased up to two orders of magnitude. The cisplatin-d(GpG) adduct affected six downstream nucleotide incorporations, but interestingly the fidelity was essentially unaltered. Biphasic kinetic analysis supported a universal kinetic mechanism for the bypass of DNA lesions catalyzed by various translesion DNA polymerases. In conclusion, if human Y-family DNA polymerases adhere to this bypass mechanism, then translesion synthesis by these error-prone enzymes is likely accountable for cisplatin resistance observed in cancer patients

Molecular Dynamics Simulations Guide Chimeragenesis and Engineered Control of Chemoselectivity in Diketopiperazine Dimerases.

In the biosynthesis of the tryptophan-linked dimeric diketopiperazines (DKPs), cytochromes P450 selectively couple DKP monomers to generate a variety of intricate and isomeric frameworks. To determine the molecular basis for selectivity of these biocatalysts we obtained a high-resolution crystal structure of selective Csp2 -N bond forming dimerase, AspB. Overlay of the AspB structure onto C-C and C-N bond forming homolog NzeB revealed no significant structural variance to explain their divergent chemoselectivities. Molecular dynamics (MD) simulations identified a region of NzeB with increased conformational flexibility relative to AspB, and interchange of this region along with a single active site mutation led to a variant that catalyzes exclusive C-N bond formation. MD simulations also suggest that intermolecular C-C or C-N bond formation results from a change in mechanism, supported experimentally through use of a substrate mimic