( 1 ) single - molecule assays were performed as described previously . additional description of protein and dna constructs , single molecule assays , data collection and analysis are given in supplementary information .
( 2 ) if dttp and atp compete for binding to helicase according to the kinetic pathway outlined in fig .
2d , then the resulting unwinding rate is : 
 vtot=(vmaxatp[atp]kmatp+vmaxdttp[dttp]kmdttp)/(1+[atp]kmatp+[dttp]kmdttp ) , where for each type of nucleotide 
 km = k1+k2k1 and vmax = sk2 with s being the step size ( in nt ) ( see supplementary discussion ) . (
3 ) in the presence of dttp and atp , if n helicase subunits coordinate in their chemo - mechanical activities and dna binding , then the resulting distance between slips ( processivity ) is : 
 dprocessivity = c(vmaxatp[atp]kmatp+vmaxdttp[dttp]kmdttp)/([atp]kmatp[atp]kmatp+[dttp]kmdttp)n1 ( supplementary discussion ) , with c being a proportionality constant .
 helicases are vital enzymes that carry out strand separation of duplex nucleic acids during replication , repair , and recombination1,2 . 
 bacteriophage t7 gene product 4 is a model hexameric helicase which has been observed to utilize dttp , but not atp , to unwind dsdna as it translocates from 5 to 3 along ssdna26 . whether and how different subunits of the helicase coordinate their chemo - mechanical activities and dna binding during translocation is still under debate1,7 . here 
 we address this question using a single molecule approach to monitor helicase unwinding . 
 we discovered that t7 helicase does in fact unwind dsdna in the presence of atp and the unwinding rate is even faster than that with dttp . 
 however unwinding traces showed a remarkable sawtooth pattern where processive unwinding was repeatedly interrupted by sudden slippage events , ultimately preventing unwinding over a substantial distance . 
 this behavior was not observed with dttp alone and was greatly reduced when atp solution was supplemented with a small amount of dttp . 
 these findings presented an opportunity to use nucleotide mixtures to investigate helicase subunit coordination . 
 we found t7 helicase binds and hydrolyzes atp and dttp by competitive kinetics such that the unwinding rate is dictated simply by their respective vmax , km , and concentrations . 
 in contrast , processivity does not follow a simple competitive behavior and shows a cooperative dependence on nucleotide concentrations . 
 this does not agree with an uncoordinated mechanism where each subunit functions independently , but supports a model where nearly all subunits coordinate their chemo - mechanical activities and dna binding . 
 our data indicate that only one subunit at a time can accept a nucleotide while other subunits are nucleotide - ligated and thus interact with the dna to ensure processivity . 
 such subunit coordination may be general to many ring - shaped helicases and reveals a potential mechanism for regulation of dna unwinding during replication . 
METHODS SUMMARY
Supplementary Material