Figure 4.

Role of ATP during assembly-driven supercoiling in yeast. (A) An extract dependent on added ATP for supercoiling (lane 1) was supplemented with adenosine 5′-[γ-thio]triphosphate (ATP-γ-S; lane 2) or adenosine 5′-[β,γ-amido]triphosphate (AMP-PNP; lane 3) instead of ATP. (B) In extracts with enough endogenous ATP to support supercoiling (lane 1), the reaction is not affected by added ATP-γ-S (lane 3) but is strongly inhibited by 3 units of apyrase (lane 2). The reactions in A and B received 100 μg of extract and were stopped after 30 min. (C) DNA ligase I (Cdc9p) functions efficiently in the context of the chromatin assembly machinery. Reactions were performed at 37°C using extracts from wild-type (lane 1) and cdc9 (lane 2) cells. Extracts were heated for 10 min at 37°C before other reaction components were added. The accumulation of nicked template in the cdc9 extract was confirmed by electrophoresis in the presence of ethidium bromide (not shown). (D) Accumulation of full-length linear DNA during assembly in the absence of ATP. The products of a standard assembly reaction are resolved by agarose gel electrophoresis in the presence of 0.25 μg/ml ethidium bromide. Open circular (O), relaxed (R), linear (L), and highly supercoiled (S) products are indicated.