PNKP and DNA ligase: Two enzymes in the polynucleotide repair pathways

Chromosomes frequently suffer damage from numerous exogenous and endogenous DNA-damaging agents. Appropriate repair of DNA damage is essential for maintaining genomic integrity and cellular viability. Polynucleotide 5' kinase and 3' phosphatase participate in DNA repair pathways by generating 5'-PO4 and 3'-OH ends to allow subsequent DNA repair synthesis and ligation. DNA ligases catalyze the ultimate step in DNA repair and replication pathways by rejoining the broken 5'-PO4/3'-OH ends. In this dissertation, I performed biochemical and structural dissection of bacteriophage polynucleotide kinase-phosphatases and two bacterial ligases.;Bifunctional polynucleotide kinase-phosphatase (PNKP) heals broken termini of RNA or DNA by converting 3'-PO4/5'-OH ends into 3'-OH/5'-PO 4 ends. PNKPs are found only in viruses and eukaryotes. Biochemical characterization of Mycobacteriophage Omega and Cjw1 PNKP indicated that viral PNKPs differ from eukaryotic PNKP with respect to quaternary structure and substrate specificity, and yet, they can substitute in vivo for the yeast end-healing components.;NAD+-dependent DNA ligase is essential for bacterial growth and is a potential target for novel antimicrobial drugs. Mutational analysis of Escherichia coli Ligase A identified residues essential for LigA activity both in vivo and in vitro. Structure-activity relationships at these essential residues were studied by conservative mutagenesis and mutational effects on individual steps of the ligation pathway were analyzed. Together with structures of Enterococcus facaelis LigA, my results help elucidate the reaction mechanism of NAD+-dependent DNA ligase, and indicate that the active site remodels as the reaction proceeds.;Bacterial Ligase D is a multifunctional enzyme composed of an ATP-dependent DNA ligase domain, a polymerase domain and a phosphoesterase (PE) domain. I showed that the ligase domain is capable of sealing DNA nicks, that the polymerase domain has templated and non-templated DNA and RNA synthesis activities, and that the PE domain has phosphodiesterase and phosphomonoesterase activities. Mutational analysis identified essential residues for all three activities and structure-function relationships at these residues provide insights to the reaction mechanisms. LigD is implicated in the bacterial NHEJ pathway. My data suggest that all three activities of LigD might be related to the NHEJ function of LigD.