Mechanism and Specificity of Human DNA Ligase I

Abstract

Cellular DNA replication and repair pathways conclude with faithful rejoining of broken phosphodiester bonds by DNA ligases. Therefore, a thorough dissection of the molecular underpinnings of DNA ligation is crucial to a comprehensive understanding of genomic maintenance. To this end, we performed biochemical and biophysical studies detailing the interactions occurring between human DNA ligase 1 (LIG1) and DNA throughout ligation. Our study led to the identification and preliminary characterization of two distinct conformational changes preceding ligation. We posit that the observed steps are distortions of the two DNA ends of the nick site, induced by LIG1 to properly align the ends for ligation. Using crystallographic data, we identified two unique sets of interactions between the DNA ends and LIG1. One such interaction is mediated through a conserved metal-binding site, which our data suggests influences the ability of LIG1 to ligate damaged or mismatched upstream ends. At the downstream end of the nick, we identified a phenylalanine residue positioned near the sugar moiety of the 5’-deoxyribose. Mutagenesis of the residue renders LIG1 unable to discriminate between a downstream deoxyribonucleotide and a downstream ribonucleotide, demonstrating its role in preventing ligation of DNA-RNA hybrids. Both mechanisms appear to influence fidelity by restricting flexibility of the downstream and upstream DNA ends. To understand how positioning of the ends contributes to ligation, we mutagenized conserved residues in the enzyme active site that were identified as putative metal ligands. Biochemical studies of the mutated residues uncovered a cooperative network of magnesium-mediated interactions between the three conserved active site residues and the DNA substrate. A biochemical characterization of two LIG1 mutations, both identified in patients suffering from broad immunological disorders, illustrates how disruption of this network of interactions can impact human health. Our study reveals that the patient-derived mutations have diminished magnesium affinity, elevating the abortive ligation burden and likely causing significant delays in DNA replication and repair. The work detailed in this thesis significantly expands our knowledge of the complex interactions between DNA ligases and their substrates, furthering our understanding of how DNA ligation fits into the larger context of DNA replication and repair.