The P1 phage replication protein RepA contacts an otherwise inaccessible thymine N3 proton by DNA distortion or base flipping

The RepA protein from bacteriophage P1 binds DNA to initiate replication. R epA covers one face of the DNA and the binding site has a completely conser ved T that directly faces RepA from the minor groove at position +7. Althou gh all four bases can be distinguished through contacts in the major groove of B-form DNA, contacts in the minor groove cannot easily distinguish betw een A and T bases. Therefore the 100% conservation at this position cannot be accounted for by direct contacts approaching into the minor groove of B- form DNA. RepA binding sites with modified base pairs at position +7 were u sed to investigate contacts with RepA. The data show that RepA contacts the N3 proton of T at position +7 and that the T=A hydrogen bonds are already broken in the DNA before RepA binds. To accommodate the N3 proton contact t he T+7 /A(+7') base pair must be distorted. One possibility is that T+7 is flipped out of the helix. The energetics of the contact allows RepA to dist inguish between all four bases, accounting for the observed high sequence c onservation. After protein binding, base pair distortion or base flipping c ould initiate DNA melting as the second step in DNA replication.