Browsing by Author "Singh, Isha"

Now showing 1 - 6 of 6
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    Building better enzymes: Molecular basis of improved non‐natural nucleobase incorporation by an evolved DNA polymerase
    (Wiley, 2020-02) Ouaray, Zahra; Singh, Isha; Georgiadis, Millie M.; Richards, Nigel G. J.; Biochemistry and Molecular Biology, School of Medicine
    Obtaining semisynthetic microorganisms that exploit the information density of “hachimoji” DNA requires access to engineered DNA polymerases. A KlenTaq variant has been reported that incorporates the “hachimoji” P:Z nucleobase pair with a similar efficiency to that seen for Watson–Crick nucleobase incorporation by the wild type (WT) KlenTaq DNA polymerase. The variant polymerase differs from WT KlenTaq by only four amino acid substitutions, none of which are located within the active site. We now report molecular dynamics (MD) simulations on a series of binary complexes aimed at elucidating the contributions of the four amino acid substitutions to altered catalytic activity. These simulations suggest that WT KlenTaq is insufficiently flexible to be able to bind AEGIS DNA correctly, leading to the loss of key protein/DNA interactions needed to position the binary complex for efficient incorporation of the “hachimoji” Z nucleobase. In addition, we test literature hypotheses about the functional roles of each amino acid substitution and provide a molecular description of how individual residue changes contribute to the improved activity of the KlenTaq variant. We demonstrate that MD simulations have a clear role to play in systematically screening DNA polymerase variants capable of incorporating different types of nonnatural nucleobases thereby limiting the number that need to be characterized by experiment. It is now possible to build DNA molecules containing nonnatural nucleobase pairs in addition to A:T and G:C. Exploiting this development in synthetic biology requires engineered DNA polymerases that can replicate nonnatural nucleobase pairs. Computational studies on a DNA polymerase variant reveal how amino acid substitutions outside of the active site yield an enzyme that replicates nonnatural nucleobase pairs with high efficiency. This work will facilitate efforts to obtain bacteria possessing an expanded genetic alphabet.
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    Crystal structures of GCN2 protein kinase C-terminal domains suggest regulatory differences in yeast and mammals
    (ASBMB, 2014-04-09) He, Hongzhen; Singh, Isha; Wek, Sheree A.; Dey, Souvik; Baird, Thomas D.; Wek, Ronald C.; Georgiadis, Millie M.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    In response to amino acid starvation, GCN2 phosphorylation of eIF2 leads to repression of general translation and initiation of gene reprogramming that facilitates adaptation to nutrient stress. GCN2 is a multidomain protein with key regulatory domains that directly monitor uncharged tRNAs which accumulate during nutrient limitation, leading to activation of this eIF2 kinase and translational control. A critical feature of regulation of this stress response kinase is its C-terminal domain (CTD). Here, we present high resolution crystal structures of murine and yeast CTDs, which guide a functional analysis of the mammalian GCN2. Despite low sequence identity, both yeast and mammalian CTDs share a core subunit structure and an unusual interdigitated dimeric form, albeit with significant differences. Disruption of the dimeric form of murine CTD led to loss of translational control by GCN2, suggesting that dimerization is critical for function as is true for yeast GCN2. However, although both CTDs bind single- and double-stranded RNA, murine GCN2 does not appear to stably associate with the ribosome, whereas yeast GCN2 does. This finding suggests that there are key regulatory differences between yeast and mammalian CTDs, which is consistent with structural differences
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    Early-Onset Pancreas Cancer: Clinical Descriptors, Genomics, and Outcomes
    (Oxford University Press, 2021) Varghese, Anna M.; Singh, Isha; Singh, Rituraj; Kunte, Siddharth; Chou, Joanne F.; Capanu, Marinela; Wong, Winston; Lowery, Maeve A.; Stadler, Zsofia K.; Salo-Mullen, Erin; Saadat, Lily V.; Wei, Alice C.; Reyngold, Marsha; Basturk, Olca; Benayed, Ryma; Mandelker, Diana; Iacobuzio-Donahue, Christine A.; Kelsen, David P.; Park, Wungki; Yu, Kenneth H.; O’Reilly, Eileen M.; Medicine, School of Medicine
    Background: Recent evidence suggests a rising incidence of cancer in younger individuals. Herein, we report the epidemiologic, pathologic, and molecular characteristics of a patient cohort with early-onset pancreas cancer (EOPC). Methods: Institutional databases were queried for demographics, treatment history, genomic results, and outcomes. Overall survival from date of diagnosis was estimated using Kaplan-Meier method. Results: Between 2008 and 2018, 450 patients with EOPC were identified at Memorial Sloan Kettering. Median overall survival was 16.3 (95% confidence interval [CI] = 14.6 to 17.7) months in the entire cohort and 11.3 (95% CI = 10.2 to 12.2) months for patients with stage IV disease at diagnosis. Of the patients, 132 (29.3% of the cohort) underwent somatic testing; 21 of 132 (15.9%) had RAS wild-type cancers with identification of several actionable alterations, including ETV6-NTRK3, TPR-NTRK1, SCLA5-NRG1, and ATP1B1-NRG1 fusions, IDH1 R132C mutation, and mismatch repair deficiency. A total of 138 patients (30.7% of the cohort) underwent germline testing; 44 of 138 (31.9%) had a pathogenic germline variant (PGV), and 27.5% harbored alterations in cancer susceptibility genes. Of patients seen between 2015 and 2018, 30 of 193 (15.5%) had a PGV. Among 138 who underwent germline testing, those with a PGV had a reduced all-cause mortality compared with patients without a PGV controlling for stage and year of diagnosis (hazard ratio = 0.42, 95% CI = 0.26 to 0.69). Conclusions: PGVs are present in a substantial minority of patients with EOPC. Actionable somatic alterations were identified frequently in EOPC, enriched in the RAS wild-type subgroup. These observations underpin the recent guidelines for universal germline testing and somatic profiling in pancreatic ductal adenocarcinoma.
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    Snapshots of an evolved DNA polymerase pre- and post-incorporation of an unnatural nucleotide
    (Oxford University Press, 2018-09-06) Singh, Isha; Laos, Roberto; Hoshika, Shuichi; Benner, Steven A.; Georgiadis, Millie M.; Biochemistry and Molecular Biology, School of Medicine
    The next challenge in synthetic biology is to be able to replicate synthetic nucleic acid sequences efficiently. The synthetic pair, 2-amino-8-(1-beta-d-2'- deoxyribofuranosyl) imidazo [1,2-a]-1,3,5-triazin-[8H]-4-one (trivially designated P) with 6-amino-3-(2'-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one (trivially designated Z), is replicated by certain Family A polymerases, albeit with lower efficiency. Through directed evolution, we identified a variant KlenTaq polymerase (M444V, P527A, D551E, E832V) that incorporates dZTP opposite P more efficiently than the wild-type enzyme. Here, we report two crystal structures of this variant KlenTaq, a post-incorporation complex that includes a template-primer with P:Z trapped in the active site (binary complex) and a pre-incorporation complex with dZTP paired to template P in the active site (ternary complex). In forming the ternary complex, the fingers domain exhibits a larger closure angle than in natural complexes but engages the template-primer and incoming dNTP through similar interactions. In the binary complex, although many of the interactions found in the natural complexes are retained, there is increased relative motion of the thumb domain. Collectively, our analyses suggest that it is the post-incorporation complex for unnatural substrates that presents a challenge to the natural enzyme and that more efficient replication of P:Z pairs requires a more flexible polymerase.
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    Structural basis for a six nucleotide genetic alphabet
    (ACS, 2015-05) Georgiadis, Millie M.; Singh, Isha; Kellet, Whitney F.; Hoshika, Shuichi; Benner, Steven A.; Richards, Nigel G. J.; Department of Chemistry & Chemical Biology, IU School of Science
    Expanded genetic systems are most likely to work with natural enzymes if the added nucleotides pair with geometries that are similar to those displayed by standard duplex DNA. Here, we present crystal structures of 16-mer duplexes showing this to be the case with two nonstandard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one) that were designed to form a Z:P pair with a standard “edge on” Watson–Crick geometry, but joined by rearranged hydrogen bond donor and acceptor groups. One duplex, with four Z:P pairs, was crystallized with a reverse transcriptase host and adopts primarily a B-form. Another contained six consecutive Z:P pairs; it crystallized without a host in an A-form. In both structures, Z:P pairs fit canonical nucleobase hydrogen-bonding parameters and known DNA helical forms. Unique features include stacking of the nitro group on Z with the adjacent nucleobase ring in the A-form duplex. In both B- and A-duplexes, major groove widths for the Z:P pairs are approximately 1 Å wider than those of comparable G:C pairs, perhaps to accommodate the large nitro group on Z. Otherwise, ZP-rich DNA had many of the same properties as CG-rich DNA, a conclusion supported by circular dichroism studies in solution. The ability of standard duplexes to accommodate multiple and consecutive Z:P pairs is consistent with the ability of natural polymerases to biosynthesize those pairs. This, in turn, implies that the GACTZP synthetic genetic system can explore the entire expanded sequence space that additional nucleotides create, a major step forward in this area of synthetic biology.
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    Structure and Biophysics for a Six Letter DNA Alphabet that Includes Imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-Diaminopyrimidine (K)
    (American Chemical Society, 2017-11-17) Singh, Isha; Kim, Myong Jung; Molt, Robert W.; Hoshika, Shuichi; Benner, Steven A.; Georgiadis, Millie M.; Biochemistry and Molecular Biology, School of Medicine
    A goal of synthetic biology is to develop new nucleobases that retain the desirable properties of natural nucleobases at the same time as expanding the genetic alphabet. The nonstandard Watson-Crick pair between imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-diaminopyrimidine (K) does exactly this, pairing via complementary arrangements of hydrogen bonding in these two nucleobases, which do not complement any natural nucleobase. Here, we report the crystal structure of a duplex DNA oligonucleotide in B-form including two consecutive X:K pairs in GATCXK DNA determined as a host-guest complex at 1.75 Å resolution. X:K pairs have significant propeller twist angles, similar to those observed for A:T pairs, and a calculated hydrogen bonding pairing energy that is weaker than that of A:T. Thus, although inclusion of X:K pairs results in a duplex DNA structure that is globally similar to that of an analogous G:C structure, the X:K pairs locally and energetically more closely resemble A:T pairs.