Mechanism of stimulation of human DNA ligase I by the Rad9-rad1-Hus1 checkpoint complex.
Accumulating evidence suggests that the Rad9-Rad1-Hus1 (9-1-1) checkpoint complex, known to be a sensor of DNA damage, is also a component of DNA repair systems. Recent results show that 9-1-1 interacts with several base excision repair proteins. It binds the DNA glycosylase MutY homolog, and stimulates DNA polymerase beta, flap endonuclease 1, and DNA ligase I. 9-1-1 resembles proliferating cell nuclear antigen (PCNA), which stimulates some of these same repair enzymes, and is loaded onto DNA in a similar manner. The complex of 9-1-1 with DNA ligase I can be immunoprecipitated from human cells. Moreover, UV irradiation stimulates 9-1-1.ligase I complex formation, suggesting a role for 9-1-1 in DNA repair. Examining the nature of 9-1-1 interaction with DNA ligase I, we show that there is a similar degree of stimulation on ligation substrates with different structures, and that there is specificity for DNA ligase I. 9-1-1 improves the binding of DNA ligase I to nicked double strand DNA. Furthermore, although high concentrations of casein kinase II strongly inhibits DNA ligase I activity, it does not affect the ability of 9-1-1 to stimulate. This suggests that 9-1-1 is also an activator of DNA ligase I during DNA damage. Unlike PCNA, 9-1-1 stimulates DNA ligase I activity to the same extent on both linear and circular substrates, indicating that encirclement is not a requirement for stimulation. These data are consistent with a direct role for 9-1-1 in DNA repair, but possibly employing a different mechanism than PCNA.
Wang
Wensheng
W
Department of Biochemistry & Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
Lindsey-Boltz
Laura A
LA
Department of Biochemistry and Biophysics, CB 7260, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599.
Sancar
Aziz
A
Department of Biochemistry and Biophysics, CB 7260, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599.
Bambara
Robert A
RA
Department of Biochemistry & Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642. Electronic address: robert_bambara@urmc.rochester.edu.
D001483:Base Sequence; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair; D005092:Exonucleases / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D029702:Schizosaccharomyces pombe Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; C000607892:LIG1 protein, human; D018809:Proliferating Cell Nuclear Antigen; D029702:Schizosaccharomyces pombe Proteins; C106332:hus1 protein, S pombe; C069930:rad9 protein; D004247:DNA; D005092:Exonucleases; C113574:Rad1 protein, human; D011088:DNA Ligases; D000072481:DNA Ligase ATP