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Figure 5.2 Méthylation protection of the EBPI binding site by EBPI

A. Hind ill to Kpn I, or Eco Ri to Pst I fragments from pSVI, 3'-end labelled at the HIndWl (top) or Eco

RI (bottom) sites, were Incubated in the absence (-) or presence (+) of 10 |xl affinity purified EBP1 and treated with DMS as described in "Materials and Methods”. DNA was cleaved at modified G residues by treatment with piperidine, reaction products were fractionated by electrophoresis in 8% denaturing polyacrylamide gels and the cleavage products visualised by autoradiography. Positions of protected bases are indicated by arrows.

B. A Bam Hi to Eco RI fragment from pUC1X72,3'-end labelled (top) or 5-end labelled (bottom), was incubated with 10 pi affinity pufiried EBPI, treated with DMS and free DNA (F) separated from EBPI - DNA complexes (B) in a native polyacrylamide gel. DNA was eluted from the gel, treated with piperidine and cleaved products fractionated by electrophoresis in 6% denaturing polyacrylamide gels. Positions of protected bases are indicated by arrows.

C. DNA sequence of the region of the SV40 enhancer containing the EBPI binding site. Filled squares represent guanines protected from DMS méthylation in the presence of EBP1 ; the open square represents a guanine hypersensitive to DMS méthylation, and the hatched square a guanine protected from DMS méthylation in pUC1X72, but not in pSVI.

245^1 244^ 240^ P S V I p U C t X 7 2

A

B

B

r

C

5'-GTTAGGGTGTGGAAAGTCCCCAGGCTC-3'

3'-CAATCCCACACCTTTCAGGGGTCCGAG-5'

I

I " ' °

I

purine ring, rendering the modified residue base labile. Cleavage of the DNA at the point of modification and analysis of the products allows us to determine which N-7s of guanine and N-3s of adenine are critical for either the formation of the DNA-protein complex, or for its maintenance. Méthylation interference experiments require that the DNA is modified such that each molecule in the population contains no more than one methylated base. The protein under study is then allowed to bind to the modified DNA and free DNA is separated from DNA-protein complexes on non-denaturing polyacrylamide gels. Modification at a site that makes an essential contact with the protein will interfere with binding; as a result that molecule will not be represented as DNA-protein complexes. DNA molecules with modifications that do not interfere with binding will be present as DNA-protein complexes. After cleavage of the DNA molecule at the point of modification, comparison of DNA present in the free and bound fractions by denaturing polyacrylamide gel electrophoresis identifies functional groups on the DNA that are essential for complex formation.

Partially methylated, [32p]-iabelled DNA representing each strand of the cioned SV1 oligonucleotide was incubated with purified EBPI and free DNA separated from DNA-protein complexes by electrophoresis in a native polyacrylamide gel. Bound and free DNA was purified, treated with NaOH, to cleave the strands at modified purine bases, and the cleavage products resolved on a sequencing gel. On the top strand the G residues at positions 240,244 and 245 that were protected from attack by DMS all interfere with binding (Figure 5.3A). Located between these interfering G residues, the A residues at positions 241, 242 and 243 also interfere with binding. On the bottom strand the two G residues at positions 236 and 237, which were protected from DMS modification, interfere with binding after modification Figure 5.3B). The two adjacent purine bases, G 238 and A 239, also interfere with binding of EBPI when modified. Guanine 235, which is hypersensitive to DMS attack and is not required for binding, does not interfere with binding when modified (data not shown). Assessment of this data indicates that the arrangement of interfering purine bases is highly strand specific; a string of four purine bases on the bottom strand followed by six purine bases on the top strand all interfere with binding (Figure 5.30). Dimethylsulphate introduces a methyl group onto the N-7 position of guanine, which projects into the major groove, and the N-3 position of adenine, which projects into the minor groove. The data presented above therefore suggest that EBPI makes contacts in both the major and minor grooves of the DNA

Figure 5.3 Methylated bases that Interfere with EBP1 binding.

Hind III to Kpn I (A) or Eco RI to Pst I (B) fragments from pSV1, 3'-end labelled at the Hind III (top) or

Eco RI (bottom) sites were treated with DMS and the methylated DNA incubated with 10 pi affinity purified EBPI. Free DNA (F) and EBP1-DNA complexes (B) were separated by electrophoresis in a native polyacrylamide gel. DNA was eluted from the gel and cleaved at modified purine bases by treatment with NaOH. Cleavage products were fractionated by electrophoresis in 8% denaturing polyacrylamide gels. Positions of interfering bases are indicated by arrows.

C. DNA sequence of the region of the SV40 enhancer containing the EBPI binding site. Filled circles depict modified bases which interfere with EBPI binding.

A p Top ° Bottom F B B P M ^G 2 3 6 ■ ^ G 2 3 7 _ ^ ^ G 2 3 8 - ^ 4 0 ^A239 c 5'-GTTAGGGTGTGGÂÂÂStCCCCAGGCTC-3' 3 ' -CAATCCCACACCTTTC^ÇÇ(JGTCCGAG-5 ' I I I

double helix.