DNA ligase I competes with FEN1 to expand repetitive DNA sequences in vitro.
Repeat sequences in various genomes undergo expansion by poorly understood mechanisms. By using an oligonucleotide system containing such repeats, we recapitulated the last steps in Okazaki fragment processing, which have been implicated in sequence expansion. A template containing either triplet or tandem repeats was annealed to a downstream primer containing complementary repeats at its 5'-end. Overlapping upstream primers, designed to strand-displace varying numbers of repeats in the downstream primer, were annealed. Human DNA ligase I joined overlapping segments of repeats generating an expansion product from the primer strands. Joining efficiency decreased with repeat length. Flap endonuclease 1 (FEN1) cleaved the displaced downstream strand and together with DNA ligase I produced non-expanded products. However, both expanded and non-expanded products formed irrespective of relative nuclease and ligase concentrations tested or enzyme addition order, suggesting the pre-existence and persistence of intermediates leading to both outcomes. FEN1 activity decreased with the length of repeat segment displaced presumably because the flap forms structures that inhibit cleavage. Increased MgCl(2) disfavored ligation of substrate intermediates that result in expansion products. Examination of expansion in vitro enables dissection of substrate and replication enzyme dynamics on repeat sequences.
Henricksen
Leigh A
LA
Department of Biochemistry and Biophysics and the Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Veeraraghavan
Janaki
J
Chafin
David R
DR
Bambara
Robert A
RA
D001483:Base Sequence; D001667:Binding, Competitive; D004247:DNA / Q000737:chemistry*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D045585:Flap Endonucleases; D006801:Humans; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D018076:DNA, Complementary; C000607892:LIG1 protein, human; D009841:Oligonucleotides; D015636:Magnesium Chloride; D004247:DNA; D005090:Exodeoxyribonucleases; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D043211:Exodeoxyribonuclease V; D011088:DNA Ligases; D000072481:DNA Ligase ATP