DNA ligases are ubiquitous enzymes that catalyze an essential final step in DNA replication and repair - the conversion of DNA nicks into phosphodiester bonds. RNA ligases participate in breakage-repair pathways that underlie tRNA splicing, post-transcriptional RNA editing, and cellular stress responses. DNA and RNA ligases seal 5'-PO4 and 3'-OH polynucleotide ends via three chemical steps: (i) ligase reacts with ATP or NAD+ to form a covalent ligase-(lysyl-N-zeta)-AMP intermediate;(ii) AMP is transferred from the ligase to the 5'-PO4 DNA or RNA strand to form a DNA/RNA-adenylate intermediate (AppDNA or AppRNA);(iii) ligase directs an attack by the 3'-OH on AppDNA/RNA to form a phosphodiester bond and release AMP.
Our aims are to understand how ligase reaction chemistry is catalyzed and how ligases recognize """"""""damaged"""""""" DNA or RNA ends. We study these problems using three model systems: a eukaryal virus-encoded DNA ligase (Chlorella virus DNA ligase: ChVLig);a bacterial NAD+-dependent DNA ligase (E. coli LigA), and a viral ATP-dependent RNA ligase (T4 Rnl2). Many physiologically important types of DNA and RNA damage result in strand breaks with 3'-PO4 or 2',3'-cyclic-PO4 (>p) ends, which cannot be sealed by classic DNA/RNA ligases. Such broken ends must be """"""""healed"""""""" - converted to a 3'-OH by a phosphoesterase - before they can be sealed. Nature has devised a remarkably diverse enzymatic tool-kit to deal with the end-healing problem. We are focused in this project on two types of end-healing systems: T4 polynucleotide kinase-phosphatase (Pnkp) and LigD phosphoesterase (LigD PE). T4 Pnkp exemplifies a large family of DNA and RNA repair proteins that convert 3'-PO4/5'-OH (or 2',3'>p/5'-OH) ends into ligatable 3'-OH/5'-PO4 ends. The mechanism of RNA cyclic-phosphate removal by T4 Pnkp is unique and entails four chemical steps and two covalent enzyme- substrate intermediates. LigD PE catalyzes two types of end-healing reactions on a DNA primer-template containing either a 3'-diribonucleotide or a 3'-PO4. The 3'-terminal nucleoside of a 3'-diribonucleotide is removed by LigD PE's phosphodiesterase activity to yield a primer strand with a ribonucleoside 3'-PO4. The 3'-PO4 is hydrolyzed by a LigD PE phosphomonoesterase activity to a 3'-OH. The atomic structure of the LigD PE domain and its active site are novel. Indeed, LigD PE defines a new superfamily of repair enzymes distributed widely in bacteria, archaea, and eukarya.
We aim to understand how T4 Pnkp and LigD PE enzymes recognize their substrates and cofactors and perform their distinctive phosphoryl transfer chemistries. We propose a multidisciplinary agenda, blending biochemistry, molecular genetics, and structural biology. Our experiments will yield new insights to phosphoryl transfer reaction mechanisms and the evolution of nucleic acid repair systems. .

Public Health Relevance

Nucleic acid end-healing and sealing enzymes are implicated in human genetic diseases. Mutations in polynucleotide kinase-phosphatase cause an autosomal recessive neurological disease characterized by microcephaly, early-onset intractable seizures, and developmental delay. Mutations in DNA ligases cause disease syndromes marked by immunodeficiency, radiation sensitivity, and developmental abnormalities. Ligases are also attractive targets for antimicrobial drug discovery. The unique substrate specificity and distinctive structure of bacterial DNA ligase (LigA) compared to human DNA ligases recommends LigA as a target for development of new broad-spectrum antibiotics. The unique structure of Rnl2-type RNA ligases highlights them as drug targets for infectious diseases caused by protozoan parasites (African sleeping sickness, Chagas disease and leishmaniasis).

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM063611-13A1
Application #
8728467
Study Section
Special Emphasis Panel (ZRG1-GGG-T (02))
Program Officer
Barski, Oleg
Project Start
2001-08-01
Project End
2018-07-31
Budget Start
2014-08-15
Budget End
2015-07-31
Support Year
13
Fiscal Year
2014
Total Cost
$262,663
Indirect Cost
$112,663
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Chauleau, Mathieu; Shuman, Stewart (2016) Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+-dependent DNA ligase (LigA). Nucleic Acids Res 44:2298-309
Chauleau, Mathieu; Shuman, Stewart (2013) Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3'-OH base mispairs and damaged base lesions. RNA 19:1840-7
Das, Ushati; Shuman, Stewart (2013) Mechanism of RNA 2',3'-cyclic phosphate end healing by T4 polynucleotide kinase-phosphatase. Nucleic Acids Res 41:355-65
Natarajan, Aswin; Dutta, Kaushik; Temel, Deniz B et al. (2012) Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D. Nucleic Acids Res 40:2076-88
Das, Ushati; Smith, Paul; Shuman, Stewart (2012) Structural insights to the metal specificity of an archaeal member of the LigD 3'-phosphoesterase DNA repair enzyme family. Nucleic Acids Res 40:828-36
Samai, Poulami; Shuman, Stewart (2012) Kinetic analysis of DNA strand joining by Chlorella virus DNA ligase and the role of nucleotidyltransferase motif VI in ligase adenylylation. J Biol Chem 287:28609-18
Smith, Paul; Nair, Pravin A; Das, Ushati et al. (2011) Structures and activities of archaeal members of the LigD 3'-phosphoesterase DNA repair enzyme superfamily. Nucleic Acids Res 39:3310-20
Samai, Poulami; Shuman, Stewart (2011) Functional dissection of the DNA interface of the nucleotidyltransferase domain of chlorella virus DNA ligase. J Biol Chem 286:13314-26
Dutta, Kaushik; Natarajan, Aswin; Nair, Pravin A et al. (2011) Sequence-specific 1H, 13C and 15N assignments of the phosphoesterase (PE) domain of Pseudomonas aeruginosa DNA ligase D (LigD). Biomol NMR Assign 5:151-5
Samai, Poulami; Shuman, Stewart (2011) Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase. J Biol Chem 286:22642-52

Showing the most recent 10 out of 44 publications