Crystal Structure of Bacteriophage T4 5′ Nuclease in Complex with a Branched DNA Reveals How Flap Endonuclease-1 Family Nucleases Bind Their Substrates

Abstract

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5 nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5 arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5 arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5 arm, lies above a magnesium binding site. The less ordered 3 arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5 arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.The flap endonuclease-1 (FEN-1) 3 nuclease family is conserved in sequence and structure from bacteriophage to humans. These nucleases play essential roles in DNA replication by removing the RNA primers from lagging strand fragments. In addition, FEN-1-related nucleases are important in long-patch base excision repair and in maintenance of genomic stability. Homozygous knockouts of FEN-1 in mice are lethal to embryos (for review, see Refs. 1-3).Like other family members, bacteriophage T4-encoded RNase H shows 5Ј to 3Ј exonuclease activity on either RNA/ DNA or DNA/DNA duplexes and endonuclease activity on either flap or fork DNA structures (4, 5). T4 rnh deletion mutants give no phage production and accumulate unligated, lagging strand fragments in Escherichia coli hosts with defective polymerase I 5Ј nuclease (6). In addition, the mutants are hypersensitive to UV irradiation and anti-tumor agents (7).FEN-1 family nuclease activities are modulated by interactions with DNA replication clamps (e.g. eukaryotic proliferating cell nuclear antigen and T4 gene 45 clamp) and single-stranded DNA-binding proteins (e.g. eukaryotic replication protein A and T4 gene 32 protein) (5, 8 -12). Human FEN-1 nuclease is also stimulated by interactions with the Werner (13, 14) and Bloom (15) syndrome helicases as well as the Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp (16).T4 RNase H 5Ј nuclease removes a short oligonucleotide (1-4 bases) each time it binds its substrate. T4 32 protein, binding on single-stranded DNA behind the nuclease, increases its processivity so that about 10 -50 short oligonucleotides are removed in a single binding event (5). On nicked sub...

The 3′-Flap Pocket of Human Flap Endonuclease 1 Is Critical for Substrate Binding and Catalysis

“…In light of the GEN activity, Chapados et al proposed that instead of threading through the helical arch, the helical arch clamps the flap structure (35). However, a recent structure of bacteriophage T4FEN1 bound to a pseudo-Y DNA shows that the 5Ј-flap appears to pass through the arch (22). Furthermore, although a portion of the T4FEN1 arch is disordered, the 5Ј-flap makes extensive contacts with arch residues.…”

“…Biochemical characterizations of FEN1 proteins from various organisms have shown that this family of nucleases can perform phosphodiesterase activity on a wide variety of substrates; however, the efficiency of catalysis on various substrates differs among the species. For instance, phage FEN1s prefer pseudo-Y substrates (22,23), whereas the archaeal and eukaryotic FEN1s prefer 5Ј-flap substrates (21,24,25), which have two dsDNA domains, one upstream and downstream of the site of cleavage, and a 5Ј-ssDNA protrusion (Fig. 1A).…”

“…Similarly, the opposite ␣-helical bundle (␣b2) has also been observed to interact with DNA (35). Based on site-directed mutagenesis studies with T5 phage FEN1 (T5FEN1) (37) and hFEN1 (38,39), and crystallographic studies of T4 phage FEN1 (T4FEN1) (22) and Archaeoglobus fulgidus FEN1 (aFEN1) (35) in complex with DNA, a general model for how FEN1 proteins recognize flap DNA has emerged. The helix-3-turn-helix motif is involved in downstream dsDNA binding, whereas the upstream dsDNA domain is bound by ␣b2.…”

“…The helix-3-turn-helix motif is involved in downstream dsDNA binding, whereas the upstream dsDNA domain is bound by ␣b2. The helical arch is likely involved in 5Ј-flap binding (22).…”

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

“…Debate about the validity or otherwise of two-metal ion catalysis has focused on several enzymes including flap endonucleases (11)(12)(13)(14)(15)(16). Crystallographic studies of substrate-free FENs have demonstrated two active site-bound metal ions (14,(17)(18)(19).…”