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J. Biol. Chem., Vol. 280, Issue 13, 12876-12887, April 1, 2005

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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^*

Omkaram Gangisetty, Charles E. Jones, Medha Bhagwat, and Nancy G. Nossal¶

From the Laboratory of Molecular and Cellular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0830

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.

Received for publication, December 14, 2004 , and in revised form, January 18, 2005.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Dept of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton St., Buffalo, NY 14263.

Present address: NCBI, National Institutes of Health, Bethesda, MD 20892.

^¶ To whom correspondence should be addressed: Laboratory of Molecular and Cellular Biology, Bldg. 8, Rm. 2A19, NIDDK, National Institutes of Health, Bethesda, MD 20892-0830. Tel.: 301-496-2724; Fax: 301-402-0240; E-mail: ngn{at}helix.nih.gov.

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