FIG. 4.

The full-site integration reaction is more sensitive to donor length than the circular half-site reaction under standard assay conditions. (A) A large quantity of the double-ended U5-U5 donor was 5′ end labeled with ³²P to produce uniformly labeled DNA. The DNA was independently digested with a variety of restriction enzymes to produce different-size donors that contain only one LTR terminus and a nonspecific end. The end-labeled fragments were separated on agarose gels and purified. A constant IN-donor end molar ratio (5 to 1) was maintained for each fragment. The concentration of IN was 80 nM, and the quantity of each single-ended fragment was varied to maintain this constant ratio. Lane 1 contains no IN with the double-ended U5-U5 donor. Lane 2 is the control U5-U5 reaction. Lanes 3 to 10 contain the single-ended U5 LTR fragments whose sizes (in base pairs) are 433, 375, 331, 290, 242, 187, 175, and 134. The restriction enzymes used to produce these different-size donors were XbaI, DrdI, BsrI, EaeI, HinfI, HinfI, EaeI, and BsrI, respectively. Nearly equivalent quantities (∼34,000 cpm) of each strand transfer reaction were subjected to agarose gel electrophoresis, and the dried gel was exposed to X-ray film. The circular half-site and full-site products along with the various-sized input donor fragments are indicated on the left. The donor-donor products migrated just above each input donor and are not marked on the side. (B) The products shown in panel A were subjected to analysis on a PhosphorImager. The percentage of donor incorporated into each product was determined as indicated on the left. The single open rectangle and triangle represent the control double-ended U5-U5 reaction, while the corresponding solid symbols represent the single-ended U5 reaction for circular half-site and full-site reactions, respectively. The sizes of the single-ended LTR fragments are indicated on the bottom.