Abstract:
DNA replication is a core biological process that rapidly occurs in both eukaryotic and prokaryotic cells with extreme precision. Gene product 32 (gp32) is a ssDNA binding protein that is important in the T4 bacteriophage DNA replication complex. Gp32 is known to bind cooperatively spanning 7 nucleotides of ssDNA. Not only is it known to bind, but it has the ability to unbind from regions of exposed ssDNA during DNA synthesis. This thesis reports microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer- template (p/t) DNA constructs of known length and polarity with and without an addition of 0.5uM gp32. The p/t constructs are characterized by two different lengths, 14-nt poly(deoxythymidine) [p(dt)14] vs 15-nt poly(deoxythymidine) [p(dt)14] and by the location that the cyanine dyes(Cy3 and Cy5) are covalently attached to the DNA at the 3’ or 5’ ends. The measurements obtained report the distance between the chromophores that are used to label the ends of 14 and 15 nucleotide segments of ssDNA attached to a p/t DNA construct. These distance measurements can track the conformational changes seen between protein bound vs. unbound states on the microsecond time scale. To analyze the data, a multipoint time correlation function analysis is utilized in order to compare the revealed kinetics of the possible conformational adaptation experienced by the ssDNA of interest. The results of our analysis demonstrate that both length and polarity of the ssDNA influence the way in which gp32 interacts with the ssDNA. The 14-nt templates have slower fluctuations than the 15-nt templates. The 5’ ssDNA constructs experience fluctuations faster than the 3’ ssDNA constructs. Therefore, this SSB is likely to play a critical role at the replication fork during DNA synthesis.