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Mechanism of DNA Binding by Human DNA Ligase 1
Author(s) -
Jurkiw Tom,
O'Brien Pat
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.753.4
Subject(s) - dna ligase , okazaki fragments , dna , dna ligases , ligation , dna clamp , dna replication , chemistry , biophysics , microbiology and biotechnology , computational biology , biochemistry , biology , eukaryotic dna replication , polymerase chain reaction , gene , reverse transcriptase
Human DNA Ligase 1 (hLIG1) is an essential enzyme in the cell that seals single‐strand DNA breaks, or nicks, that occur between adjacent Okazaki Fragments during DNA replication. Although the phosphoryl transfer steps of hLIG1 have been previously characterized, many questions remain about how hLIG1 is able find and recognize nick sites. New approaches are needed to observe the rapid and transient interactions. Here we have investigated the kinetic mechanism for DNA binding using fluorescence approaches. By using a nicked DNA reporter and endogenous tryptophan fluorescence, we are able to monitor the DNA‐dependent steps of the ligation mechanism. Under the conditions used the assay appears to report on initial interactions with the nicked DNA substrate and the product release step, both of which are previously uncharacterized steps. Future experiments will extend these kinetic and thermodynamic studies to understand what features of hLIG1 and the nicked DNA substrate allow for rapid and faithful ligation of DNA breaks. Support or Funding Information Research was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number T32GM008353 and the Rackham Graduate School and Biological Chemistry Department at the University of Michigan.