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.
1. Growth and UV Irradiation.
2. DNA Isolation.
3. 2D Gel and Southern Analysis.
4. Representative Results:
Figure 1. Plasmid replication intermediates observed in the presence and absence of UV-induced DNA damage. The migration pattern of PvuII digested pBR322 plasmid observed by 2D-agarose-gel analysis is diagrammed. Non-replicating linear plasmids run as a linear 4.4-kb fragment. Replicating plasmids form Y-shaped structures that migrate slower than the non-replicating fragments due to their larger size and nonlinear shape. This migration pattern forms an arc that extends out from the linear region towards the well. Following UV-irradiation, double-Y or X-shaped molecules are observed in the cone region that migrates more slowly than the arc of Y-shaped molecules. An example of the Southern analysis of the 2D gel probed with 32P-labeled pBR322 is shown for cells immediately after UV irradiation and 15 minutes after UV irradiation (reproduced with permission from the publisher).
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 repaired.
In place of the Southern analysis, the replication intermediates can be punched out of the gel with a plastic drinking straw, purified, and observed directly by electron microscopy. We have used this approach successfully to identify gene products required to process replication forks that encounter DNA damage and to identify abnormal replication intermediates that accumulate in these mutants . In addition, this approach can be easily modified to examine other forms of DNA damage.
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 |