SV40 large T antigen helicase: Roles of the hydrophilic channels and a newly identified unwinding activity

To initiate DNA replication, SV40 large T antigen needs to recognize and bind to the origin in SV40 DNA. This is achieved through sequence specific contacts between the T antigen origin binding domain (OBD) and the pentanucleotide regions in the center of the origin, as well as non-sequence specific interactions between the helicase domain and the EP and AT regions of the origin. This leads to the assembly of a double hexamer of T antigen over a melted origin DNA. Subsequently, the DNA becomes unwound bidirectionally, providing SS DNA templates for DNA replication. However, these events are not fully understood. For instance, it is not clear how T antigen separates DS DNA at the origin or how the separated strands become threaded through the double hexamer. In this dissertation, I identified a new unwinding activity of large T antigen. The helicase can unwind the central pentanucleotides in an ATP-hydrolysis dependent way. This activity of T antigen, like its structural distortion activity that acts on the flanking EP and AT regions, is an indispensable component of the unwinding of the entire origin. Specific sequences in the pentanucleotide region are necessary for the activity, and the flanking AT track can stimulate it.;The SV40 hexameric helicase consists of a positively charged central channel and six hydrophilic channels in the large tiers. The hydrophilic channels separate adjacent large tier domains within each hexamer. In this dissertation, I generated a series of mutants with single-point substitutions in the hydrophilic channels and studied the function of the hydrophilic channels in SV40 DNA replication. The mutants were characterized in various biochemical assays and grouped into four major classes according to their properties.;One class of mutant proteins (class A-D429A, N449S and N515S) displayed normal DNA binding, ATPase, and helicase activities. However, they were defective specifically at unwinding origin DNA. The three mutants were found to be deficient in EP melting activity. Two of them (D429A and N515S) also failed to unwind the central pentanucleotide region of the origin. Taken together, my data indicate that the hydrophilic channels play active roles in unwinding the origin to provide the single-stranded substrates for DNA replication.;Based on my results and on published structural and functional information, a model is proposed in this dissertation to illustrate how T antigen could separate the pentanucleotide region at the center of the origin. My data also suggest a mechanism by which the step-wise assembly of the T antigen double hexamer could trigger unwinding of the origin.