We present a procedure by which two-dimensional agarose-gel analysis can be used to identify the structure of replication intermediates that occur following UV irradiation.
Typical results obtained from wild type cells in the presence and absence of UV-induced damage are shown in Figure 1. In the absence of damage, ~1% of the total plasmid DNA can be found in the Y arc when cells are rapidly growing in exponential phase. Following irradiation, a transient increase in Y shaped molecules is observed as blocked replication forks accumulate at damaged sites. The X-shaped replication intermediates also transiently accumulate and persist until a time that correlates with when the lesions are rep…
The authors have nothing to disclose.
Work in our lab is supported by CAREER award MCB0551798 from the National Science Foundation and AREA grant R15GM86839 from the NIGMS-NIH.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
An example of the Southern analysis of the 2D gel probed | GE | G15T8 | Yellow Lighting | |
15 μwatt germicidal lamp | Sylvania | F20T12/GO | UV Lamp | |
Blak-Ray UV Intensity Meter 254nm | Daigger | EF28195T | UVC photometer | |
0.025 μm pore disks | Whatman | VSWP04700 | Floating dialysis disks | |
PvuII | Fermentas | ER0632 | Restriction Endonuclease | |
Nick-translation kit | Roche Diagnostics | 976776 | To make 32P-labeled probe | |
Blotting Paper | Whatman | 3030-704 | For Southern transfer | |
Nylon membrane | GE Healthcare | RPN203S | For Southern transfer |