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Primer delivery by T7 gene 4 proteins
Author(s) -
Zhu Bin,
Lee SeungJoo,
Richardson Charles C.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.648.3
Subject(s) - primase , replisome , primer (cosmetics) , dna polymerase , biology , dna replication , dna , microbiology and biotechnology , helicase , gene , dnag , genetics , biochemistry , polymerase chain reaction , chemistry , circular bacterial chromosome , reverse transcriptase , organic chemistry , rna
Gene 4 protein (gp4) encoded by bacteriophage T7 contains a C‐terminal helicase domain and a N‐terminal primase domain. After synthesis of the tetraribonucleotide primers gp4 must then transfer them to the polymerase for use as primers to initiate DNA synthesis. A truncated version of the full‐length 63‐kDa gp4, the 56‐kDa gp4, lacks the N‐terminal Cys 4 zinc‐binding motif that is important in the recognition of primase sites in the DNA. We find that 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. This property of the 56‐kDa gp4 is potentially significant in the initiation of T7 DNA replication. We have identified DNA primases that are defective in primer delivery by screening 56‐kDa gp4s containing mutations for their ability to support the growth of T7 phage lacking gene 4 but containing a suppressor that allows it to grow in the presence of the 56‐kDa gp4. We find that Trp69 of the 56‐kDa gp4 is critical for primer delivery. Substitution of lysine for Trp69 yields 56‐kDa gp4‐W69K and 63‐kDa gp4‐W69K that are defective in primer delivery to the polymerase with other functions unaffected. DNA primase harboring lysine at position 69 fails to stabilize the primer on DNA as analyzed by surface plasmon resonance.