Gene 5.5 protein of bacteriophage T7 in complex with Escherichia coli nucleoid protein H-NS and transfer RNA masks transfer RNA priming in T7 DNA replication

Abstract

DNA primases provide oligoribonucleotides for DNA polymerase to initiate lagging strand synthesis. A deficiency in the primase of bacteriophage T7 to synthesize primers can be overcome by genetic alterations that decrease the expression of T7 gene 5.5, suggesting an alternative mechanism to prime DNA synthesis. The product of gene 5.5 (gp5.5) forms a stable complex with the Escherichia coli histone-like protein H-NS and transfer RNAs (tRNAs). The 3′-terminal sequence (5′-ACCA-3′) of tRNAs is identical to that of a functional primer synthesized by T7 primase. Mutations in T7 that suppress the inability of primase reduce the amount of gp5.5 and thus increase the pool of tRNA to serve as primers. Alterations in T7 gene 3 facilitate tRNA priming by reducing its endonuclease activity that cleaves at the tRNA-DNA junction. The tRNA bound to gp5.5 recruits H-NS. H-NS alone inhibits reactions involved in DNA replication, but the binding to gp5.5-tRNA complex abolishes this inhibition.A 3′-hydroxyl end of an RNA or DNA strand positioned at the catalytic site of DNA polymerase functions as a primer to initiate synthesis of DNA. In most instances, primers are short oligoribonucleotides synthesized by enzymes designated DNA primases (1). DNA primases catalyze the template-directed synthesis of short oligoribonucleotides and stabilize the newly synthesized primer on the template and hand it off to DNA polymerase for initiation of DNA synthesis (2, 3).Bacteriophage T7 has provided a model system for the study of DNA replication. Only three T7 proteins-gene 5 DNA polymerase (gp5), gene 4 primase-helicase (gp4), and gene 2.5 ssDNAbinding protein (gp2.5)-together with host Escherichia coli thioredoxin, are sufficient for leading and lagging strands synthesis in a reconstituted replication system (4). The genes encoding these proteins, together with genes 2 (E. coli RNA polymerase inhibitor), 3 (endonuclease), 3.5 (lysozyme), and 6 (exonuclease) constitute the DNA metabolism class II genes. More than a dozen additional genes within the class II group remain uncharacterized (5).The primase activity of T7 is located in the N-terminal half of the 63-kDa gp4, whereas the C-terminal half of gene 4 encodes the DNA helicase (4). Like other prokaryotic homologs, the primase domain recognizes a specific sequence (1, 6) 5′-(G/T)(G/T) GTC-3′ in the ssDNA template to catalyze the template-directed synthesis of functional tetraribonucleotides (pppACCA, pppACCC, and pppACAC) that the lagging strand DNA polymerase can use as primers to initiate the synthesis of Okazaki fragments. Gene 4 encodes two colinear proteins, the full-length 63-kDa gp4 and a short 56-kDa form in equal amount in vivo from an internal start codon and ribosome-binding site (4). This 56-kDa gp4 has full helicase activity but lacks the N-terminal zinc-binding domain (ZBD), an indispensable motif for primer synthesis (7). Despite its inability to synthesize template-dependent tetraribonucleotides, the 56-kDa gp4 is capable of stabilizing certain preformed oligonucleotides on ...

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

“…Even more intriguing is our recent finding that the T7 DNA primase, under circumstances where it cannot itself synthesize RNA primers, can use preformed oligonucleotides and even tRNA as primers for lagging strand DNA synthesis (24). In this reaction, 3Ј-sequences in the oligonucleotides complementary to those in the primase recognition sequence are annealed to the primase recognition sequence and are extended by T7 DNA polymerase.…”

“…Hydrolysis of RNA-terminated Flaps by gp6-We recently reported that under certain circumstances, preformed oligonucleotides and even tRNA could serve as primers for T7 DNA polymerase in conjunction with the 56-kDa T7 gene 4 primase/ helicase (24). The functional oligoribonucleotides synthesized by the T7 DNA primase are pppACCC, pppACCA, and pppACAC.…”

“…This result indicates that gp3 and not gp6 could play a role in the removal of tRNA primers. gp3 endonuclease has been implicated in the use of tRNA as primer in T7 phage-infected cells (24).…”

“…However, a number of circumstances can arise in T7-infected cells where flap endonuclease activity may play a critical role. We recently reported that the primase domain of the 56-kDa T7 gene 4 protein can use preformed oligonucleotides and even tRNA as functional primers for T7 DNA polymerase (24). In this reaction, the primase anneals the preformed primer (the 3Ј-end of tRNA) to the primase recognition site provided the four terminal 3Ј-nucleotides of the preformed RNA fragment are complementary to those of the recognition site.…”

It Seems Like Only Yesterday

“…H-NS binds to double-stranded DNA, particularly bent or curved DNA; condenses the DNA; and results in global gene regulation (283). Almost 20 years, later Bin Zhu and colleagues in my lab (240) identified mutations in gene 5.5 that allowed T7 phage, which lacked the ability to synthesize RNA primers, to nevertheless grow in E. coli. Subsequent studies revealed that gene 5.5 protein binds to E. coli transfer RNA (tRNA) and that this complex sequesters the H-NS protein.…”