Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5 0 -OH and 3 0 -phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.

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Expression of human lecithin cholesterol acyltransferase (LCAT) in mice (LCAT-Tg) leads to increased high density lipoprotein (HDL) cholesterol levels but paradoxically, enhanced atherosclerosis. We have hypothesized that the absence of cholesteryl ester transfer protein (CETP) in LCAT-Tg mice facilitates the accumulation of dysfunctional HDL leading to impaired reverse cholesterol transport and the development of a pro-atherogenic state. To test this hypothesis we crossbred LCAT-Tg with CETP-Tg mice. On both regular chow and high fat, high cholesterol diets, expression of CETP in LCAT-Tg mice reduced total cholesterol (؊39% and ؊13%, respectively; p < 0.05), reflecting a decrease in HDL cholesterol levels. CETP normalized both the plasma clearance of Increased plasma levels of high density lipoproteins (HDL) 1 are powerful indicators of low cardiovascular risk in humans (1-3). This relationship may in part be indirect, reflecting the fact that high levels of HDL are a marker for efficient clearance of pro-atherogenic remnant particles from the circulation (4). However, increased plasma HDL levels achieved by either infusion of HDL or overexpression of the apoA-I gene (5-8) protect against the development of atherosclerosis in different animal models, indicating a direct anti-atherogenic role of HDL. The mechanism underlying this relationship is poorly understood. Potential anti-atherogenic properties of HDL include antioxidant effects (9, 10) as well as the ability to prevent monocyte recruitment into the intima (11, 12), to inhibit the aggregation of atherogenic lipoproteins (13, 14), and to serve as a thrombolytic agent (15). HDL has also been proposed to play a major role in reverse cholesterol transport, a process that involves the movement of cholesterol from peripheral cells to the liver for removal from the body (16 -19). Thus, raising plasma HDL by dietary, pharmacologic, or genetic interventions may be an effective strategy for the prevention of cardiovascular disease in humans.Cholesteryl ester transfer protein (CETP) and lecithin cholesterol acyltransferase (LCAT) are two key proteins that modulate the plasma concentrations of HDL. LCAT mediates the esterification of free cholesterol on plasma lipoproteins, thereby converting discoidal, nascent HDL particles into mature spherical HDL containing a central core of cholesteryl esters (CE) (16,19). Overexpression of LCAT in transgenic rabbits (20, 21) increases the plasma HDL cholesterol levels and significantly reduces aortic atherosclerosis. LCAT transgenic mice (LCAT-Tg) also have elevated HDL cholesterol levels (22-24). However, high plasma HDL concentrations are associated with either no change (25) or enhanced diet-induced atherosclerosis in LCAT-Tg mice (26).CETP is another key protein that modulates the plasma levels of HDL. CETP promotes the transfer of CE from HDL to apoB-containing lipoproteins in exchange for triglyceride (19,(27)(28)(29). The absence of atherosclerosis in some of the first CETP-deficient Japanese patients to be described (30...

Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier

Small GTPase proteins such as Ras are key regulators of cellular proliferation and are activated by guanine nucleotide exchange/releasing factors (GEFs/GRFs). Three classes of Ras GRFs have been identified to date, represented by Sos1/2, Ras-GRF1/2 and Ras-GRP. Here, we describe a novel candidate Ras activator, cyclic nucleotide rasGEF (CNrasGEF), which contains CDC25, Ras exchange motif (REM), Ras-association (RA), PDZ and cNMP (cAMP/cGMP) binding (cNMP-BD) domains, two PY motifs and a carboxy-terminal SxV sequence. CNrasGEF can activate Ras in vitro, and it binds cAMP directly via its cNMP-BD. In cells, CNrasGEF activates Ras in response to elevation of intracellular cAMP or cGMP, or treatment with their analogues 8-Br-cAMP or 8-Br-cGMP, independently of protein kinases A and G (PKA and PKG). This activation is prevented in CNrasGEF lacking its CDC25 domain or cNMP-BD. CNrasGEF can also activate the small GTPase Rap1 in cells, but this activation is constitutive and independent of cAMP. CNrasGEF is expressed mainly in the brain and is localized at the plasma membrane, a localization dependent on the presence of intact PDZ domain but not the SxV sequence. These results suggest that CNrasGEF may directly connect cAMP-generating pathways or cGMP-generating pathways to Ras.

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Poly(ADP-ribosyl)ation by poly(ADP-ribose) polymerases regulates the interaction of many DNA damage and repair factors with sites of DNA strand lesions. The interaction of these factors with poly (ADP-ribose) (PAR) is mediated by specific domains, including the recently identified PAR-binding zinc finger (PBZ) domain. However, the mechanism governing these interactions is unclear. To better understand the PBZ-PAR interaction, we performed a detailed examination of the representative PBZ-containing protein involved in the DNA damage response, aprataxin polynucleotide-kinase-like factor (APLF), which possesses two tandem PBZ domains. Here we present structural and biochemical studies that identify Y381/Y386 and Y423/Y428 residues in the conserved C(M/P)Y and CYR motifs within each APLF PBZ domain that are critical for the interaction with the adenine ring of ADP-ribose. Basic residues (R387 and R429 in the first and second PBZ domains, respectively) coordinate additional interactions with the phosphate backbone of ADP-ribose, suggesting that APLF binds to multiple ADP-ribose residues along PAR polymers. These C(M/P)Y and CYR motifs form a basic/hydrophobic pocket within a variant zinc finger structure and are required for APLF recruitment to sites of DNA damage in vivo.DNA damage signaling | high affinity T he DNA damage response (DDR) and maintenance of chromosomal stability is regulated in part by posttranslational modifications, including poly(ADP-ribosyl)ation by poly(ADPribose) polymerases (PARPs), which direct the recruitment of proteins involved in the signaling and repair of DNA damage. In addition, PARP enzymes are important regulators of chromatin remodeling, apoptosis, and transcription (1). In the early response to DNA damage, PARP1 is the predominant PARP activated by DNA strand lesions and catalyzes the attachment of multiple ADP-ribose (ADPr) units from nicotinamide adenine dinucleotide (NAD þ ) onto target proteins, including PARP1 itself (2). Poly(ADP-ribose) (PAR) accumulated at DNA breaks is subsequently metabolized to ADPr by PAR glycohydrolase (3). To date, PAR-binding motifs have been described in some macrodomains, which also bind to ADPr (4, 5), in a basic residue-rich motif interspersed with hydrophobic amino acids (6), and in the more recently described PAR-binding zinc finger (PBZ) domains (7). PBZ domains, present in proteins either as single or two tandem motifs, are limited to multicellular eukaryotes, and the majority of PBZ-containing proteins have putative roles in PAR metabolism, DNA repair, or DNA damage signaling (7-9).APLF (aprataxin polynucleotide kinase (PNK)-like factor, also known as PALF and Xip1) is a newly identified protein involved in the DDR possessing a forkhead-associated (FHA) domain and two tandem PBZ motifs (7-11) (Fig. 1A). APLF was originally identified based on the similarity of its FHA domain to those of PNK and aprataxin (12), which share functional similarities and direct FHA-and phosphothreonine-dependent interactions with the DNA repair proteins XRCC1 and...