Heterohexamer of 56- and 63-kDa gene 4 helicase-primase of bacteriophage T7 in DNA replication

Huidong Zhang, Seung Joo Lee, Arkadiusz W. Kulczyk, Bin Zhu, Charles C. Richardson

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Bacteriophage T7 expresses two forms of gene 4 protein (gp4). The 63-kDa full-length gp4 contains both the helicase and primase domains. T7 phage also express a 56-kDa truncated gp4 lacking the zinc binding domain of the primase; the protein has helicase activity but no DNA-dependent primase activity. Although T7 phage grow better when both forms are present, the role of the 56-kDa gp4 is unknown. The two molecular weight forms oligomerize by virtue of the helicase domain to form heterohexamers. The 56-kDa gp4 and any mixture of 56- and 63-kDa gp4 show higher helicase activity in DNA unwinding and strand-displacement DNA synthesis than that observed for the 63-kDa gp4. However, single-molecule measurements show that heterohexamers have helicase activity similar to the 63-kDa gp4 hexamers. In oligomerization assays the 56-kDa gp4 and any mixture of the 56- and 63-kDa gp4 oligomerize to form more hexamers than does the 63-kDa gp4. The zinc binding domain of the 63-kDa gp4 interferes with hexamer formation, an inhibition that is relieved by the insertion of the 56-kDa species. Compared with the 63-kDa gp4, heterohexamers synthesize a reduced amount of oligoribonucleotides, mediated predominately by the 63-kDa subunits via a cis mode. During coordinated DNA synthesis 7% of the tetraribonucleotides synthesized are used as primers by both heterohexamers and hexamers of the 63-kDa gp4. Overall, an equimolar mixture of the two forms of gp4 shows the highest rate of DNA synthesis during coordinated DNA synthesis.

Original languageEnglish (US)
Pages (from-to)34273-34287
Number of pages15
JournalJournal of Biological Chemistry
Volume287
Issue number41
DOIs
StatePublished - Oct 5 2012

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DNA Primase
Bacteriophage T7
Bacteriophages
DNA Replication
Genes
DNA
Zinc
Oligoribonucleotides
Oligomerization
Proteins
Molecular Weight
Assays
Molecular weight
Molecules

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Cite this

Zhang, Huidong ; Lee, Seung Joo ; Kulczyk, Arkadiusz W. ; Zhu, Bin ; Richardson, Charles C. / Heterohexamer of 56- and 63-kDa gene 4 helicase-primase of bacteriophage T7 in DNA replication. In: Journal of Biological Chemistry. 2012 ; Vol. 287, No. 41. pp. 34273-34287.
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abstract = "Bacteriophage T7 expresses two forms of gene 4 protein (gp4). The 63-kDa full-length gp4 contains both the helicase and primase domains. T7 phage also express a 56-kDa truncated gp4 lacking the zinc binding domain of the primase; the protein has helicase activity but no DNA-dependent primase activity. Although T7 phage grow better when both forms are present, the role of the 56-kDa gp4 is unknown. The two molecular weight forms oligomerize by virtue of the helicase domain to form heterohexamers. The 56-kDa gp4 and any mixture of 56- and 63-kDa gp4 show higher helicase activity in DNA unwinding and strand-displacement DNA synthesis than that observed for the 63-kDa gp4. However, single-molecule measurements show that heterohexamers have helicase activity similar to the 63-kDa gp4 hexamers. In oligomerization assays the 56-kDa gp4 and any mixture of the 56- and 63-kDa gp4 oligomerize to form more hexamers than does the 63-kDa gp4. The zinc binding domain of the 63-kDa gp4 interferes with hexamer formation, an inhibition that is relieved by the insertion of the 56-kDa species. Compared with the 63-kDa gp4, heterohexamers synthesize a reduced amount of oligoribonucleotides, mediated predominately by the 63-kDa subunits via a cis mode. During coordinated DNA synthesis 7{\%} of the tetraribonucleotides synthesized are used as primers by both heterohexamers and hexamers of the 63-kDa gp4. Overall, an equimolar mixture of the two forms of gp4 shows the highest rate of DNA synthesis during coordinated DNA synthesis.",
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Heterohexamer of 56- and 63-kDa gene 4 helicase-primase of bacteriophage T7 in DNA replication. / Zhang, Huidong; Lee, Seung Joo; Kulczyk, Arkadiusz W.; Zhu, Bin; Richardson, Charles C.

In: Journal of Biological Chemistry, Vol. 287, No. 41, 05.10.2012, p. 34273-34287.

Research output: Contribution to journalArticle

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AU - Richardson, Charles C.

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