Introducing a single pyrimidine dimer into a DNA sequence

[Scott Keeney]

In article <3jd2g7$29g at ixnews4.ix.netcom.com>,
Andrew Akerman <Akman at ix.netcom.com> wrote:
>	Hi, I'm a student doing  project on DNA repair. I am interested 
>in analyzing the binding affinities of several repair protiens to a 
>pyrimidine dimer (caused by UV irradiation.) I have constructed a very 
>elegant experiment involving gel mobility-shift assay, but I require a 
>double stranded DNA sequence with only a single dimer in it (preferably 
>in the center.) I was hoping that maybe someone can direct me to a source 
>of literature where I can get help to build this "template." Any help is 
>greatly appreciated. Thank you.
>
>					A. Akerman
>Email address: Akman at ix.netcom.com


Check out the paper by Treiber, Chen, and Essigmann (nucleic acids
research 20(21): 5805-5810).  They made synthetic double-stranded oligos
with a central oligopyrimidine tract of either 5 T's or 4 T's and a C.
When UV-irradiated, the T5 oligo formed cyclobutane pyrimidine dimers, 
while the T4C oligo formed a mix of cyclobutanes and (6,4) photoproducts.

You might also want to check out Reardon et al., 1993 (J. Biological 
Chemistry 268:21301-21308).  This paper used chemically synthesized
lesion-containing oligonucleotides to measure binding affinities
of the human xeroderma pigmentosum group E DNA damage binding protein
and the E. coli UvrA protein using a gel shift assay. 

There are a host of other relevant references, but those are the only two
I have handy at the moment.

Hope this helps.

scott keeney
dept of molecular nad cellular biology
harvard university