Maturation of Bacteriophage T4 Lagging Strand Fragments Depends on Interaction of T4 RNase H with T4 32 Protein Rather than the T4 Gene 45 Clamp

Charles E Jones,Omkaram Gangisetty,Nancy G Nossal,Medha Bhagwat

doi:10.1074/jbc.m414025200

Abstract

In the bacteriophage T4 DNA replication system, T4 RNase H removes the RNA primers and some adjacent DNA before the lagging strand fragments are ligated. This 5'-nuclease has strong structural and functional similarity to the FEN1 nuclease family. We have shown previously that T4 32 protein binds DNA behind the nuclease and increases its processivity. Here we show that T4 RNase H with a C-terminal deletion (residues 278-305) retains its exonuclease activity but is no longer affected by 32 protein. T4 gene 45 replication clamp stimulates T4 RNase H on nicked or gapped substrates, where it can be loaded behind the nuclease, but does not increase its processivity. An N-terminal deletion (residues 2-10) of a conserved clamp interaction motif eliminates stimulation by the clamp. In the crystal structure of T4 RNase H, the binding sites for the clamp at the N terminus and for 32 protein at the C terminus are located close together, away from the catalytic site of the enzyme. By using mutant T4 RNase H with deletions in the binding site for either the clamp or 32 protein, we show that it is the interaction of T4 RNase H with 32 protein, rather than the clamp, that most affects the maturation of lagging strand fragments in the T4 replication system in vitro and T4 phage production in vivo.

Highlights

Ases with conserved sequences and similar structures (Fig. 1)
Our studies indicate that the mechanism by which lagging strand polymerization and primer removal are coordinated in bacteriophage T4 replication is different from that used in eukaryotes
T4 RNase H Exonuclease Activity Is Stimulated When the T4 Gene 45 Clamp Protein Is Loaded Behind It—The T4 gene 45 replication clamp protein is loaded preferentially at the 3Ј end of a junction between single- and double-stranded DNA and at the 3Ј side of a nick [11, 12], whereas T4 RNase H is loaded at the 5Ј end [3]

Summary

EXPERIMENTAL PROCEDURES

DNA Substrates—Oligonucleotides were made and reverse phasepurified by Sigma-Genosys, except that oligonucleotides longer than 50 bases were gel-purified. T4 Replication Proteins—Wild type T4 RNase H, T4 DNA polymerase, T4 gene 45 clamp, genes 44/62 clamp loader, gene 41 helicase, gene 59 helicase loading protein, and gene 61 primase were purified to apparent homogeneity as described by Nossal et al [20]. Primase, helicase, RNase H, and DNA ligase were incubated for 2 min at 37 °C, and synthesis was begun by the addition of polymerase, primase, and helicase. Point mutations in the N-terminal region of T4 RNase H were made by site-directed mutagenesis of the wild type gene in the plasmid pVC2001 [3], using the method of Kunkel et al [25], modified by using T4 DNA polymerase, T4 44/64 clamp loader, and 45 clamp to copy the ssDNA template. Total phage were measured at 60 min, after lysis with chloroform

RESULTS

DISCUSSION

Methods