Jayati Roy Choudhury

Mount Sinai School of Medicine, Manhattan, NY, United States

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Publications (10)58.92 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The biological functions of human DNA polymerase (Pol) theta, an A family Pol, have remained poorly defined. Here we identify a role of Pol theta in translesion synthesis (TLS) in human cells. We show that TLS through the thymine glycol (Tg) lesion, the most common oxidation product of thymine, occurs via two alternative pathways, in one of which, Pols kappa and zeta function together and mediate error-free TLS, whereas in the other, Pol theta functions in an error-prone manner. Human Pol theta is comprised of an N-terminal ATPase/helicase domain, a large central domain, and a C-terminal polymerase domain; however, we find that only the C-terminal polymerase domain is required for TLS opposite Tg in human cells. In contrast to TLS mediated by Pol kappa and Pol zeta, in which Pol zeta would elongate the chain from the Tg:A base pair formed by Pol kappa action, the ability of Pol theta alone to carry out the nucleotide insertion step, as well as the subsequent extension step which presents a considerable impediment due to displacement of the 5' template base, suggests that the Pol theta active site can accommodate highly distorting DNA lesions.
    Journal of Biological Chemistry 03/2014; · 4.60 Impact Factor
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    ABSTRACT: DNA polymerase ζ (Polζ) is specialized for the extension step of translesion DNA synthesis (TLS). Despite its central role in maintaining genome integrity, little is known about its overall architecture. Initially identified as a heterodimer of the catalytic subunit Rev3 and the accessory subunit Rev7, yeast Polζ has recently been shown to form a stable four-subunit enzyme (Polζ-d) upon the incorporation of Pol31 and Pol32, the accessory subunits of yeast Polδ. To understand the 3D architecture and assembly of Polζ and Polζ-d, we employed electron microscopy. We show here how the catalytic and accessory subunits of Polζ and Polζ-d are organized relative to each other. In particular, we show that Polζ-d has a bilobal architecture resembling the replicative polymerases and that Pol32 lies in proximity to Rev7. Collectively, our study provides views of Polζ and Polζ-d and a structural framework for understanding their roles in DNA damage bypass.
    Cell Reports 10/2013; · 7.21 Impact Factor
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    ABSTRACT: A major clinical problem in the use of cisplatin to treat cancers is tumor resistance. DNA polymerase η (Pol-η) is a crucial polymerase that allows cancer cells to cope with the cisplatin-DNA adducts that are formed during chemotherapy. We present here a structure of human Pol-η inserting deoxycytidine triphosphate (dCTP) opposite a cisplatin intrastrand cross-link (PtGpG). We show that the specificity of human Pol-η for PtGpG derives from an active site that is open to permit Watson-Crick geometry of the nascent PtGpG-dCTP base pair and to accommodate the lesion without steric hindrance. This specificity is augmented by the residues Gln38 and Ser62, which interact with PtGpG, and Arg61, which interacts with the incoming dCTP. Collectively, the structure provides a basis for understanding how Pol-η in human cells can tolerate the DNA damage caused by cisplatin chemotherapy and offers a framework for the design of inhibitors in cancer therapy.
    Nature Structural & Molecular Biology 05/2012; 19(6):628-32. · 11.63 Impact Factor
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    Jayati Roy Choudhury, Lu Rao, Ulrich Bierbach
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    ABSTRACT: A restriction enzyme cleavage inhibition assay was designed to determine the rates of DNA platination by four non-cross-linking platinum-acridine agents represented by the formula [Pt(am(2))LCl](NO(3))(2), where am is a diamine nonleaving group and L is an acridine derived from the intercalator 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (ACRAMTU). The formation of monofunctional adducts in the target sequence 5'-CGA was studied in a 40-base-pair probe containing the EcoRI restriction site GAATTC. The time dependence of endonuclease inhibition was quantitatively analyzed by polyacrylamide gel electrophoresis. The formation of monoadducts is approximately 3 times faster with double-stranded DNA than with simple nucleic acid fragments. Compound 1 (am(2) is ethane-1,2-diamine, L is ACRAMTU) reacts with a first-order rate constant of k (obs) = 1.4 ± 0.37 × 10(-4) s(-1) (t (1/2) = 83 ± 22 min). Replacement of the thiourea group in ACRAMTU with an amidine group (compound 2) accelerates the rate by fourfold (k (obs) = 5.7 ± 0.58 × 10(-4) s(-1), t (1/2) = 21 ± 2 min), and introduction of a propane-1,3-diamine nonleaving group results in a 1.5-fold enhancement in reactivity (compound 3, k (obs) = 2.1 ± 0.40 × 10(-4) s(-1), t (1/2) = 55 ± 10 min) compared with the prototype. Derivative 4, containing a 4,9-disubstituted acridine threading intercalator, was the least reactive compound in the series (k (obs) = 1.1 ± 0.40 × 10(-4) s(-1), t (1/2) = 104 ± 38 min). The data suggest a correlation may exist between the binding rates and the biological activity of the compounds. Potential pharmacological advantages of rapid formation of cytotoxic monofunctional adducts over the common purine-purine cross-links are discussed.
    European Journal of Biochemistry 11/2010; 16(3):373-80. · 3.16 Impact Factor
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    ABSTRACT: Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase kappa (Polkappa) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polkappa's preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polkappa in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide. We show that the Polkappa active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP. The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polkappa is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polkappa is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases.
    PLoS ONE 02/2009; 4(6):e5766. · 3.53 Impact Factor
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    ABSTRACT: Background: Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major lesions formed. It is amongst the most mutagenic lesions in cells because of its dual coding potential, wherein 8-oxoG(syn) can pair with an A in addition to normal base pairing of 8-oxoG(anti) with a C. Human DNA polymerase κ (Polκ) is a member of the newly discovered Y-family of DNA polymerases that possess the ability to replicate through DNA lesions. To understand the basis of Polκ's preference for insertion of an A opposite 8-oxoG lesion, we have solved the structure of Polκ in ternary complex with a template-primer presenting 8-oxoG in the active site and with dATP as the incoming nucleotide. Methodology and Principal Findings: We show that the Polκ active site is well-adapted to accommodate 8-oxoG in the syn conformation. That is, the polymerase and the bound template-primer are almost identical in their conformations to that in the ternary complex with undamaged DNA. There is no steric hindrance to accommodating 8-oxoG in the syn conformation for Hoogsteen base-paring with incoming dATP. Conclusions and Significance: The structure we present here is the first for a eukaryotic translesion synthesis (TLS) DNA polymerase with an 8-oxoG:A base pair in the active site. The structure shows why Polκ is more efficient at inserting an A opposite the 8-oxoG lesion than a C. The structure also provides a basis for why Polκ is more efficient at inserting an A opposite the lesion than other Y-family DNA polymerases.
    PLoS ONE 01/2009; 4(6). · 3.53 Impact Factor
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    ABSTRACT: The cytotoxic complex, [PtCl(Am)2(ACRAMTU)](NO3)2 (1) ((Am)2 = ethane-1,2-diamine, en; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea), is a dual platinating/intercalating DNA binder that, unlike clinical platinum agents, does not induce DNA cross-links. Here, we demonstrate that substitution of the thiourea with an amidine group leads to greatly enhanced cytotoxicity in H460 non-small-cell lung cancer (NSCLC) in vitro and in vivo. Two complexes were synthesized: 4a (Am2 = en) and 4b (Am = NH3), in which N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine replaces ACRAMTU. Complex 4a proves to be a more efficient DNA binder than complex 1 and induces adducts in sequences not targeted by the prototype. Complexes 4a and 4b induce H460 cell kill with IC(50) values of 28 and 26 nM, respectively, and 4b slows tumor growth in a H460 mouse xenograft study by 40% when administered at a dose of 0.5 mg/kg. Compound 4b is the first non-cross-linking platinum agent endowed with promising activity in NSCLC.
    Journal of Medicinal Chemistry 12/2008; 51(23):7574-80. · 5.48 Impact Factor
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    ABSTRACT: Four highly charged, water soluble platinum-acridine bisintercalating agents have been synthesized. Depending on the cis/trans isomerism of the metal and the nature of the acridine side chains, bisintercalation induces/stabilizes the classical Watson-Crick B-form or a non-B-form. Circular dichroism spectra and chemical footprinting experiments suggest that 4, the most active derivative in HL-60 cells, produces a structurally severely perturbed DNA with features of a Hoogsteen base-paired biopolymer.
    Journal of Medicinal Chemistry 07/2008; 51(11):3069-72. · 5.48 Impact Factor
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    ABSTRACT: The following complexes of type [PtCl(R)(ACRAMTU)](NO3)2 (ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea)), derived from prototype 1 (with R = ethane-1,2-diamine), were synthesized: 2 (with R = (1R,2R)-1,2-diaminocyclohexane), 3 (with R = propane-1,3-diamine), 4 (with R = N1,N1,N2,N2-tetramethylethane-1,2-diamine), and 5 (with R = 2,2'-bipyridine). The DNA sequence specificity of the conjugates and their antiproliferative potential in HL-60 and H460 cells were investigated. Conjugate 3 showed the strongest non-cisplatin-type DNA damage in polymerase stop assays and superior cell kill efficacy in H460 lung cancer (IC50 = 70 nM).
    Journal of Medicinal Chemistry 06/2007; 50(9):2259-63. · 5.48 Impact Factor
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    Jayati Roy Choudhury, Ulrich Bierbach
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    ABSTRACT: The DNA interactions of PT-BIS(ACRAMTU) ([Pt(en)(ACRAMTU)2](NO3)4; ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea, en = ethylenediamine), a bifunctional platinum-acridine conjugate, have been studied in native and synthetic double-stranded DNAs and model duplexes using various biophysical techniques. These include ethidium-DNA fluorescence quenching and thermal melting experiments, circular dichroism (CD) spectroscopy and plasmid unwinding assays. In addition, the binding mode was studied in a short octamer by NMR spectroscopy in conjunction with molecular modeling. In alternating copolymers, PT-BIS(ACRAMTU) shows a distinct preference for poly(dA-dT)2, which is approximately 3-fold higher than that of ACRAMTU. In the ligand-oligomer complex, d(GCTATAGC)2.PT-BIS(ACRAMTU) (complex I*), PT-BIS(ACRAMTU) increases the thermal stability of the B-form host duplex by DeltaT(m) > 30 K (CD and UV melting experiments). The agent unwinds pSP73 plasmid DNA by 44(+/-2) degrees per bound molecule, indicating bisintercalative binding. A 2-D NMR study unequivocally demonstrates that PT-BIS(ACRAMTU)'s chromophores deeply bisintercalate into the 5'-TA/TA base pair steps in I*, while the platinum linker lies in the minor groove. An AMBER model reflecting the NMR results shows that bracketing of the central AT base pairs in a classical nearest neighbor excluded fashion is feasible. PT-BIS(ACRAMTU) inhibits DNA hydrolysis by BstZ17 I at the enzyme's restriction site, GTA downward arrowTAC. Possible consequences for other relevant DNA-protein interactions, such as those involved in TATA-box-mediated transcription initiation and the utility of the platinum-intercalator technology for the design of sequence-specific agents are discussed.
    Nucleic Acids Research 01/2005; 33(17):5622-32. · 8.81 Impact Factor

Publication Stats

120 Citations
58.92 Total Impact Points

Institutions

  • 2012
    • Mount Sinai School of Medicine
      • Department of Structural and Chemical Biology
      Manhattan, NY, United States
  • 2005–2010
    • Wake Forest University
      • Department of Chemistry
      Winston-Salem, North Carolina, United States
  • 2009
    • University of Texas Medical Branch at Galveston
      • Department of Biochemistry and Molecular Biology
      Galveston, TX, United States