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).

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-GGG-T (02))
Program Officer
Barski, Oleg
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Sloan-Kettering Institute for Cancer Research
New York
United States
Zip Code
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
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
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
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
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
Zhu, Hui; Shuman, Stewart (2010) Gap filling activities of Pseudomonas DNA ligase D (LigD) polymerase and functional interactions of LigD with the DNA end-binding Ku protein. J Biol Chem 285:4815-25
Nair, Pravin A; Smith, Paul; Shuman, Stewart (2010) Structure of bacterial LigD 3'-phosphoesterase unveils a DNA repair superfamily. Proc Natl Acad Sci U S A 107:12822-7

Showing the most recent 10 out of 42 publications