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E for examining the interface between the helicase and primase domains of gene 4 protein was the obvious conclusion that any communication must pass through or involve this region. For example, even though the twoJOURNAL OF BIOLOGICAL CHEMISTRYInterface of T7 Helicase-Primasedomains cannot part into solution, transient dissociations and reassociations of the two domains could still occur. Our results suggest that contacts at the interface of helicase and primase can mediate this regulation. Single amino acid changes in helix E of this region result in changes in the primase and helicase activities that mimic events observed during coordinated DNA replication in the T7 system. For example, the K408A alteration enhances primase but inhibits helicase activity, simultaneous events observed during primer synthesis using single-molecule techniques (7). Consistently, gp4-K408A has a slightly higher affinity for ssDNA compared with wildtype gene 4 protein (Fig. 3). Interestingly, gp4-K408D differs from gp4-K408A in that it has significantly less primase activity, clearly showing that relatively minor alterations in helix E can regulate primase activity. Although gp4-K408A appears to be in a "priming mode," gp4-G415V is in an "unwinding" mode where helicase activity is activated and primase activity diminished. It is noteworthy that gp4-G415V does not oligomerize as well as wild-type protein, suggesting that a relatively loose conformation favors helicase activity. In contrast, the priming mode might arise from a more stable oligomer, as shown by gp4-K408A bound to DNA. Residues Lys408, Tyr411, and Gly415 are on the same side of helix E, facing the primase domain. Residue Arg404, also on this face, plays an essential role. Alterations of Arg404 lead to the loss of in vivo function as well as an inability to unwind DNA. The crystal structure of gene 4 protein does not reveal an interaction with another residue. However, more conformational change, for instance, induced by ssDNA binding could make it feasible for these residues to interact. In contrast, most alterations of residues located in helix E but not on the same surface as the four residues mentioned above do not lead to severe defects in vivo. From our data it appears that Asp263, located in the linker of one subunit, interacts with Tyr411 in helix E in the hexamer and with https://britishrestaurantawards.org/members/redcandle3/activity/372588/ Lys408 in helix E of the heptamer and with Arg404 in helix E. Thus the switch in contacts between Asp263 and the interacting residues in helix E of the adjacent subunit of the oligomeric gene 4 protein provides conformational changes to modulate helicase and primase activity. A model to explain the effects of alterations in helix E on helicase and primase activity is presented in Fig. 7. During DNA replication the gene 4 hexamer exists in a relaxed conformation with the primase domains splayed out from the helicase domain (Fig. 7C). In this conformation residue Asp263 of one subunit contacts Lys408 and/or Arg404 in helix E of the adjacent subunit. Primase activity is silenced as the helicase domain unwinds DNA for the leading strand DNA polymerase. gp4-G415V mimics this conformational state. Gly415 contacts an unknown part of the primase domain. Increasing the size of the side chain of Gly415 might also increase the distance between the neighboring residues Tyr411 and Asp263 in the linker. Consequently, the G415V alteration favors contacts between Asp263 and Lys408 and/or Arg404 to maintain the relaxed conformation of.