Abstract
Ku, also known as nuclear Factor IV, is an abundant nuclear DNA-binding protein which requires free DNA ends for the initial interaction with double-stranded DNA (dsDNA) and can bind at multiple sites along dsDNA in an energy-independent manner. Its function in vivo is unknown, but it has been implicated in both DNA replication and repair and in transcriptional control. We have used an electrophoretic mobility shift assay to further define the DNA binding properties of the Ku protein. Titration of Ku to a fixed amount of any of several target linear dsDNA fragments produced ladders of shifted bands proportional to the length of DNA, confirming the multiple binding activity of Ku and demonstrating its sequence-independent nature. Using a short DNA fragment with one Ku binding site, the binding constant of Ku for dsDNA ends was calculated to be 2.4 x 10(9) M-1. Competitive inhibition experiments confirmed the requirement of a free DNA end for binding by Ku and demonstrated that Ku binds isolated nicks in dsDNA. Nick binding was also observed directly using radiolabeled singly nicked circular DNA. The relative affinities of Ku for specific nick sites and free DNA ends were approximately equal, and nick binding was sequence-independent. Finally, in a study of a possible role for Ku in protecting or repairing damaged DNA, Ku was shown to inhibit the ability of T4 DNA ligase to circularize linear dsDNA molecules, demonstrating that some Ku molecules remain at the DNA terminus rather than translocate. A similar inhibition was not observed at nicks. These experiments document a new DNA binding specificity for Ku and further suggest that the high affinity end and nick binding activity is biologically relevant to its functions in vivo.
Highlights
From the Section of Rheumatology, DeDartment of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510
Doublets were CAMP-binding protein (CAP) and the lac repressor(Fried seen in our earlier studiesof the interaction of radiola- andCrothers, 1981, 1984), andthatdetermined by timebeled, in uitro synthesized Ku protein with DNA (Griffith et resolved fluorescence spectroscopy for the Klenow fragment al., 1992a)
Since the initialdescription of the Ku autoantigenin 1981 (Mimori et al, 1981), much has been learned about its structure and properties (Allaway et al, 1990; Chan et al, 1989; Griffith et al, 1992a; Mimori and Hardin,1986; Mimori et al, 1986; Reeves and Sthoeger, 1989; Yaneva et al, 1989)
Summary
From the Section of Rheumatology, DeDartment of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510. Ends was calculated to be 2.4 X los M-’ Competitive These properties, aswell as its abundance,lack of sequence inhibition experiments confirmed the requiremoefnat specificity, and affinity for DNA ends, have suggested a role free DNA end for binding Kbuyand demonstrated that for Ku in basic DNA metabolic processes such as replication, Ku binds isolated nicks in dsDNA. Ina study of a possible role foKr u in protecting RNA(Knuth et al, 1990), andanoctamerconsensusseor repairing damaged DNA, Ku was shown to inhibit quence-binding protein (Mayet al., 1991) Those observations theability of T4 DNA ligasetocircularizelinear suggest that Ku may have a role in transcription. Oursystem usesa competitive electrophoretic mobility shift assay to study the affinity of Ku for various DNA substrates In these experiments, we haveobtainedfurther evidence thatKubinds multiple sites on DNA after binding to ends, in an energyandsequence-independentmanner. The abbreviations used are: NFIV, nuclear factor IV; bp, base pair(s); ds, double-stranded; ss, single-stranded; EthBr, ethidium bromide
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