Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Proc Natl Acad Sci U S A. 2010 May 18;107(20):9099-104. doi: 10.1073/pnas.1004220107. Epub 2010 May 3.
Gene 4 protein (gp4) encoded by bacteriophage T7 contains a C-terminal helicase and an N-terminal primase domain. After synthesis of tetraribonucleotides, gp4 must transfer them to the polymerase for use as primers to initiate DNA synthesis. In vivo gp4 exists in two molecular weight forms, a 56-kDa form and the full-length 63-kDa form. The 56-kDa gp4 lacks the N-terminal Cys(4) zinc-binding motif important in the recognition of primase sites in DNA. The 56-kDa gp4 is defective in primer synthesis but delivers a wider range of primers to initiate DNA synthesis compared to the 63-kDa gp4. Suppressors exist that enable the 56-kDa gp4 to support the growth of T7 phage lacking gene 4 (T7Delta4). We have identified 56-kDa DNA primases defective in primer delivery by screening for their ability to support growth of T7Delta4 phage in the presence of this suppressor. Trp69 is critical for primer delivery. Replacement of Trp69 with lysine in either the 56- or 63-kDa gp4 results in defective primer delivery with other functions unaffected. DNA primase harboring lysine at position 69 fails to stabilize the primer on DNA. Thus, a primase subdomain not directly involved in primer synthesis is involved in primer delivery. The stabilization of the primer by DNA primase is necessary for DNA polymerase to initiate synthesis.
噬菌体 T7 编码的基因 4 蛋白 (gp4) 包含一个 C 端解旋酶和一个 N 端引物酶结构域。在合成四核苷酸后，gp4 必须将其转移到聚合酶上，用作启动 DNA 合成的引物。在体内，gp4 存在两种分子量形式，一种是 56kDa 形式，另一种是全长 63kDa 形式。56kDa 的 gp4 缺乏 N 端 Cys(4)锌结合基序，该基序对于在 DNA 中识别引物酶位点很重要。与 63kDa 的 gp4 相比，56kDa 的 gp4 在引物合成中存在缺陷，但能提供更广泛的引物来启动 DNA 合成。存在一些抑制剂，使 56kDa 的 gp4 能够支持缺乏基因 4 的 T7 噬菌体（T7Delta4）的生长。我们通过筛选其在这种抑制剂存在下支持 T7Delta4 噬菌体生长的能力，鉴定出 56kDa DNA 引物酶在引物传递方面存在缺陷。色氨酸 69 对引物传递至关重要。在 56-或 63kDa 的 gp4 中，将色氨酸替换为赖氨酸会导致引物传递缺陷，而其他功能不受影响。在位置 69 处具有赖氨酸的 DNA 引物酶无法稳定引物在 DNA 上。因此，一个不直接参与引物合成的引物酶亚结构域参与了引物传递。DNA 引物酶稳定引物对于 DNA 聚合酶启动合成是必要的。
