Gregory P. Mullen

Solution structure of the single-strand break repair protein XRCC1 N-terminal domain.

XRCC1 functions in the repair of single-strand DNA breaks in mammalian cells and forms a repair complex with β-Pol, ligase III and PARP. Here we describe the NMR solution structure of the XRCC1 N-terminal domain (XRCC1 NTD). The structural core is a β-sandwich with β-strands connected by loops, three helices and two short two-stranded β-sheets at each connection side. We show, for the first time, that the XRCC1 NTD specifically binds single-strand break DNA (gapped and nicked). We also show that the XRCC1 NTD binds a gapped DNA–β-Pol complex. The DNA binding and β-Pol binding surfaces were mapped by NMR and found to be well suited for interaction with single-strand gap DNA containing a 90° bend, and for simultaneously making contacts with the palm-thumb of β-Pol in a ternary complex. The findings suggest a mechanism for preferential binding of the XRCC1 NTD to flexible single-strand break DNA.

Solution structure of a dynein motor domain associated light chain.

Dyneins are molecular motors that translocate towards the minus ends of microtubules. In Chlamydomonas flagellar outer arm dynein, light chain 1 (LC1) associates with the nucleotide binding region within the γ heavy chain motor domain and consists of a central leucine-rich repeat section that folds as a cylindrical right handed spiral formed from six β-β-α motifs. This central cylinder is flanked by terminal helical subdomains. The C-terminal helical domain juts out from the cylinder and is adjacent to a hydrophobic surface within the repeat region that is proposed to interact with the dynein heavy chain. The position of the C-terminal domain on LC1 and the unexpected structural similarity between LC1 and U2A′ from the human spliceosome suggest that this domain interacts with the dynein motor domain.

Structural basis for the topological specificity function of MinE.

Correct positioning of the division septum in Escherichia coli depends on the coordinated action of the MinC, MinD and MinE proteins. Topological specificity is conferred on the MinCD division inhibitor by MinE, which counters MinCD activity only in the vicinity of the preferred midcell division site. Here we report the structure of the homodimeric topological specificity domain of Escherichia coli MinE and show that it forms a novel αβ sandwich. Structure-directed mutagenesis of conserved surface residues has enabled us to identify a spatially restricted site on the surface of the protein that is critical for the topological specificity function of MinE.

Solution structure of a viral DNA repair polymerase.

DNA polymerase X (Pol X) from the African swine fever virus (ASFV) specifically binds intermediates in the single-nucleotide base-excision repair process, an activity indicative of repair function. In addition, Pol X catalyzes DNA polymerization with low nucleotide-insertion fidelity. The structural mechanisms by which DNA polymerases confer high or low fidelity in DNA polymerization remain to be elucidated. The three-dimensional structure of Pol X has been determined. Unlike other DNA polymerases, Pol X is formed from only a palm and a C-terminal subdomain. Pol X has a novel palm subdomain fold, containing a positively charged helix at the DNA binding surface. Purine deoxynucleoside triphosphate (dNTP) substrates bind between the palm and C-terminal subdomain, at a dNTP-binding helix, and induce a unique conformation in Pol X. The purine dNTP–bound conformation and high binding affinity for dGTP–Mg2+ of Pol X may contribute to its low fidelity.

Mapping of the Interaction Interface of DNA Polymerase β with XRCC1

Abstract Residues of DNA polymerase β (β-Pol) that interact with the DNA repair protein XRCC1 have been determined by NMR chemical shift mapping (CSM) and mutagenesis. 15 N/ 13 C/ 2 H/ 1 H, 13 C-methyl Leu,Ile,Val -labeled β-Pol palm-thumb domain was used for assignments of the 1 H, 15 N, and 13 C resonances used for CSM of the palm-thumb on forming the 40 kDa complex with the XRCC1 N-terminal domain (NTD). Large chemical shift changes were observed in the thumb on complexation. 15 N relaxation data indicate reduction in high-frequency motion for a thumb loop and three palm turn/loops, which showed concomitant chemical shift changes on complexation. A ΔV303–V306 deletion and an L301R/V303R/V306R triple mutation abolished complex formation due to loss in hydrophobicity. In an updated model, the thumb-loop of β-Pol contacts an edge/face region of the β sheet of the XRCC1 NTD, while the β-Pol palm weakly contacts the α2 helix.

The dimerization and topological specificity functions of MinE reside in a structurally autonomous C-terminal domain

Correct placement of the division septum in Escherichia coli requires the co‐ordinated action of three proteins, MinC, MinD and MinE. MinC and MinD interact to form a non‐specific division inhibitor that blocks septation at all potential division sites. MinE is able to antagonize MinCD in a topologically sensitive manner, as it restricts MinCD activity to the unwanted division sites at the cell poles. Here, we show that the topological specificity function of MinE residues in a structurally autonomous, trypsin‐resistant domain comprising residues 31–88. Nuclear magnetic resonance (NMR) and circular dichroic spectroscopy indicate that this domain includes both α and β secondary structure, while analytical ultracentrifugation reveals that it also contains a region responsible for MinE homodimerization. While trypsin digestion indicates that the anti‐MinCD domain of MinE (residues 1–22) does not form a tightly folded structural domain, NMR analysis of a peptide corresponding to MinE1–22 indicates that this region forms a nascent helix in which the peptide rapidly interconverts between disordered (random coil) and α‐helical conformations. This suggests that the N‐terminal region of MinE may be poised to adopt an α‐helical conformation when it interacts with the target of its anti‐MinCD activity, presumably MinD.

Insights into the mechanism of the β-elimination catalyzed by the N-terminal domain of DNA polymerase β

Abstract The N-terminal domain of DNA polymerase β carries an activity for excision of deoxyribose 5-phosphate from DNA at an interaction interface that includes a helix-hairpin-helix motif containing Lys-68 and Lys-72 and an adjacent Ω loop containing His-34 and Lys-35. His-34 displays a low pK a (5.7) due to proximity to Lys-35. In a proposed mechanism, Lys-68 protonates the hemiacetal O4′ and His-34 stabilizes the protonated aldehyde-type species, possibly accepting the proton. In one of two possible mechanisms, Lys-68 or Lys-72 forms a Schiffs base with the substrate. His-34 can function in C2′ deprotonation for the Lys-68 Schiffs base intermediate, while the proton would be transferred to water for the Lys-72 Schiffs base. Lys-35 stabilizes the phosphomonoester leaving group in either case.

Letter to the Editor: 1H, 15N, and 13C resonance assignments for the N-terminal 20 kDa domain of the DNA single-strand break repair protein XRCC1

XRCC1 is a 633-residue protein necessary for the repair of single-strand DNA breaks in mammalian cells. The XRCC1 protein has three apparent domains that include the N-terminal 20 kDa domain, a central BRCT domain, and a C-terminal BRCT domain. Two intervening segments of the XRCC1 protein have not been classified. Chinese hamster ovary cell lines with mutations in the XRCC1 gene have characteristic DNA repair defects that include sensitivity to ionizing radiation, elevated levels of single-strand DNA breaks, and increased sister chromatid exchange (Thompson et al., 1990; Shen et al., 1998). Using methods that included affinity chromatography and yeast two-hybrid screening, the XRCC1 N-terminal domain and intact XRCC1 were shown to interact with mammalian β-Pol (Kubota et al., 1996). The C-terminal BRCT domain interacts with a BRCT domain in DNA ligase III. The central BRCT domain of XRCC1 has been shown to interact with a BRCT domain on PARP (Masson et al., 1998). Thus, the XRCC1 protein has been considered to be a scaffolding protein, necessary for formation of a βPol-XRCC1-DNA ligase III repair complex, and this complex may also interact with PARP. No structural information is available for the XRCC1 N-terminal domain. Here we report on the 1H, 15N, and 13C res∗To whom correspondence should be addressed. E-mail: gmullen@panda.uchc.edu Abbreviations: XRCC1, X-ray cross-complementing group 1 protein; β-Pol, DNA polymerase β; BER, base excision repair; BRCT, BRCA1 (breast cancer susceptibility protein) C-terminal domain; PARP, poly(ADP-ribose) polymerase; IPTG, isopropyl β-D-thiogalactopyranoside; AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride; DTT, dithiothreitol. onance assignments for the backbone and essentially all side chains within the XRCC1 20 kDa N-terminal domain.

Letter to the Editor: 1H, 15N, and 13C resonance assignments for a 20 kDa DNA polymerase from African swine fever virus

The African Swine Fever Virus (ASFV) is known to cause a lethal disease in domestic pigs. The virus contains two DNA polymerases; a eukaryotic-like DNA polymerase involved in viral DNA replication and a 174 amino acid DNA polymerase that is a member of the polymerase X family (Pol X). This family includes DNA polymerase β (β-Pol), terminal transferase (TdT), DNA polymerase μ (Pol μ) (Dominguez et al., 2000), and DNA polymeraseλ (Pol λ) (Aoufouchi et al., 2000). One notable difference between Pol X and the Pol X family is the absence of N-terminal DNA binding domains that are found in β-Pol, TdT, Pol μ, and Pol λ and the absence of BRCT domains that are found in Pol μ and Pol λ. In comparison to β-Pol, Pol X comprises only the palm and C-terminal subdomains. Based on biochemical analysis, Pol X was shown to be similar to β-Pol with respect to template-directed DNA synthesis, preference for deoxynucleotides versus ribonucleotides, and repair of a single nucleotide gap DNA (Oliveros et al., 1997). This would suggest that Pol X acts in base excision repair of the viral DNA inside the cytoplasm of the host cell. In addition, Pol X catalyzes the formation of a G:G mismatch with relatively high catalytic efficiency (Showalter and Tsai, 2001) and may provide mutase function to ASFV. As the smallest DNA polymerase, Pol X represents the minimal core needed for a protein to act as a template directed nucleotidal transferase. Here we report on the nearly complete backbone and side chain 1H, 15N, and 13C resonance assignments for the 20 kDa Pol X.