Backbone dynamics and refined solution structure of the N-terminal domain of DNA polymerase β. Correlation with DNA binding and dRP lyase activity1

Article No. jmbi.1999.3455 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 296, 229±253

Backbone Dynamics and Refined Solution Structure of the N-terminal Domain of DNA Polymerase b . Correlation with DNA Binding and dRP Lyase Activity Mark W. Maciejewski1, Dingjiang Liu1, Rajendra Prasad2, Samuel H. Wilson2 and Gregory P. Mullen1* 1

Department of Biochemistry University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032, USA 2

National Institute of Environmental Health Sciences 111 T. W. Alexander Drive Building 101, Research Triangle Park, NC 27709, USA

Mammalian DNA polymerase b functions in the base excision DNA repair pathway ®lling in short patches (1-5 nt) in damaged DNA and removing deoxyribose 50 -phosphate from the 50 -side of damaged DNA. The backbone dynamics and the re®ned solution structure of the N-terminal domain of b-Pol have been characterized in order to establish the potential contribution(s) of backbone motion to the DNA binding and deoxyribose 50 -phosphate lyase function of this domain. The N-terminal domain is formed from four helices packed as two antiparallel pairs with a 60  crossing between the pairs. The RMSD of the NMR conformers Ê for the backbone heavy atoms and 0.78 A Ê for (residues 13-80) is 0.37 A all heavy atoms. NMR characterization of the binding site(s) for a ssDNA-5mer, ssDNA-8mer, ssDNA-9mer, and dsDNA-12mer shows a consensus surface for the binding of these various DNA oligomers, that surrounds and includes the deoxyribose 50 -phosphate lyase active site region. Connection segments between helices 1 and 2 and between helices 3 and 4 each contribute to DNA binding. Helix-3-turn-helix-4 forms a helix-hairpin-helix motif. The highly conserved hairpin sequence (LPGVG) displays a signi®cant degree of picosecond time-scale motion within the backbone, that is possibly important for DNA binding at the phosphodiester backbone. An -loop connecting helices 1 and 2 and helix-2 itself display signi®cant exchange contributions (Rex) at the backbone amides due to apparent conformational type motion on a millisecond time-scale. This motion is likely important in allowing the -loop and helix-2 to shift toward, and productively interact with, gapped DNA. The deoxyribose 50 -phosphate lyase catalytic residues that include K72 which forms the Schiff's base, Y39 which is postulated to promote proton transfer to the aldehyde, and K35 which assists in phosphate elimination, show highly restricted backbone motion. H34, which apparently participates in detection of the abasic site hole and assists in the opening of the hemiacetal, shows conformational exchange. # 2000 Academic Press

15

*Corresponding author

Keywords: DNA polymerase b; N relaxation; protein dynamics; conformational exchange; DNA binding

Present address: Dingjian Liu, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA. Abbreviations used: nt, nucleotide; dRP, deoxyribose 50 -phosphate; b-Pol, DNA polymerase b; BER, base excision repair; HhH, helix-hairpin-helix; CSI, chemical shift index; R1, 15N translational relaxation rate; R2, 15N transverse relaxation rate; S2, order parameter; te, effective internal correlation time; Rex, exchange contribution to R2; J(o), spectral density function; NLS, nuclear localization sequence; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; NOESY-HSQC, nuclear Overhauser enhancement spectroscopy-heteronuclear single quantum coherence; HNCA, 1Hi-15Ni-13Cai correlated 3D NMR; HN(CO)CA, 1Hi-15Ni-13Caiÿ1 correlated 30 NMR; DQF-COSY, doublequantum ®ltered correlated spectroscopy; HMQC-J, heteronuclear multiple-quantum coherence J-resolved; NOE, nuclear Overhauser enhancement. E-mail address of the corresponding author: [email protected] 0022-2836/00/60229±25 $35.00/0

# 2000 Academic Press

230

Introduction DNA is a molecule that is prone to chemical modi®cation, and it is estimated that many thousands of DNA modi®cations occur within a cell per day. Preservation of the genetic code depends on repair of DNA damage. Mammalian DNA polymerase b (b-Pol) functions in the base excision repair (BER) pathway and in maintaining a stable genome in humans. Mutant forms of b-Pol are associated with defects in DNA repair and cancer cell lines (Bhattachary & Banerjee, 1997). In BER, repair enzymes catalyze the hydrolysis of the glycosidic bond at nucleotides containing damaged bases and removal of the abasic sugar-phosphate. In the BER pathway, b-Pol catalyzes DNA polymerization and deoxyribose 50 -phosphate excision at incised abasic sites in double-stranded DNA (dsDNA). Abasic sites are generated by speci®c DNA glycosylases or by spontaneous purine hydrolysis. AP endonuclease catalyzes phosphodiester bond hydrolysis at the 50 -side of the abasic site (i.e. incision) and provides the free 30 -OH group that is necessary for polymerization. Concomitant with this step, the AP endonuclease participates in loading b-Pol at the abasic site (Bennett et al., 1997). Kinetic evidence indicates that b-Pol next catalyzes nucleotide polymerization (Srivastava et al., 1998). In the penultimate step in BER, b-Pol catalyzes removal of the deoxyribose 50 -phosphate (dRP) group and thereby provides a required phosphate group at the 50 terminus for DNA ligation. The dRP lyase active site is located on the 8 kDa N-terminal domain of b-Pol. In addition to providing dRP lyase activity, the b-Pol N-terminal domain functions in gapped dsDNA binding and in single-stranded DNA (ssDNA) binding. The solution structure and a ssDNA interaction for the b-Pol N-terminal domain have been determined (Liu et al., 1994, 1996). A structural interaction by the b-Pol N-terminal domain at a singlenucleotide gap in DNA has been characterized (Sawaya et al., 1997). The b-Pol N-terminal domain is composed of four helices packed as two approximately antiparallel pairs. Helices 3 and 4 of the domain form what is termed a helix-hairpin-helix (HhH) motif, a highly conserved motif found in several BER enzymes. In the b-Pol N-terminal domain, the HhH forms a signi®cant portion of the DNA interaction surface. HhH motifs have been structurally characterized in the Escherichia coli endonuclease III (Kuo et al., 1992) and the 3-MeA DNA glycosylase (Labahn et al., 1996; Yamagata et al., 1996) in the absence of DNA. The apparent function of the HhH includes phosphate-backbone binding (Doherty et al., 1996) and recognition of damaged DNA (Mullen & Wilson, 1997). In the b-Pol N-terminal domain, additional DNA contacts are made by residues in and adjacent to an -loop connecting helices 1 and 2. The dRP-lyase active site is formed by a group of residues that are a subset of the DNA binding surface (Prasad et al.,

Backbone Dynamics of the -Pol N-terminal Domain

1998). Residues, K72, K35, and Y39 form part of a shallow pocket, and K72 and K35 have been shown to directly contribute to catalysis. Residues, H34 and K60 are outside the dRP lyase pocket, but also contribute to dRP lyase catalysis (Prasad et al., 1998). A mechanistic scheme for dRP catalysis has been proposed (Scheme 1), that is consistent with the mutagenesis results for these residues (Mullen et al., 1997). In the original solution structure of the b-Pol Nterminal domain (Liu et al., 1996), two segments were found to contact ssDNA and to display a high backbone heavy atom RMSD within the group of NMR conformers. This led us to a complete study of the backbone dynamics presented here. In view of the dynamics results, we have also completed a re®nement of the solution structure of the b-Pol N-terminal domain. We here assess the potential role that backbone dynamics plays in both DNA interaction and in the dRP lyase activity exhibited by the b-Pol N-terminal domain. Two approaches have been used to assess motional contributions to relaxation. In one approach, the model-free formalism (Lipari & Szabo, 1982a,b) was used to ®t the experimentally measured relaxation rates and NOE values using a spectral density function describing rotation of the protein and model-free internal motion. In the other approach, termed reduced spectral density mapping, the relaxation rates and NOE values were used to directly determine the spectral density function at a reduced set of frequencies (Farrow et al., 1995; Ishima & Nagayama, 1995a,b; Lefevere et al., 1996).

Results Refined solution structure The 533 NOE restraints and 68 dihedral angles from the original solution structure were supplemented with 388 additional meaningful NOE restraints and 67 CSI derived backbone dihedral angle restraints in determining the re®ned solution structure (Table 1). The re®ned solution structure is shown in Figure 1(a), and the structural statistics are shown in Table 1. The ensemble of 25 structures form a tight bundle for residues 13-80 with Ê for backbone and an RMSD of 0.37(0.05) A Ê 0.78(0.04) A for all heavy atoms. The RMSD values for backbone and heavy atoms for segments within the N-terminal domain and comparisons to the original solution structure are given in Table 2. The improvement in precision has led to an approximate 50 % reduction in RMSD values of all regions except the -loop where the improvement was more dramatic with the RMSD reduced to Ê from 1.63(0.73) A Ê . As is described 0.32(0.05) A below, the RMSD values for each of the secondary structural elements correspond well with ¯exibility as determined by 1H-15N nuclear Ovehauser enhancement (NOE) values, order paramter (S2), effect correlation time (te), and spectral density function (hJ(oH)i). The previous solution structure

231

Backbone Dynamics of the -Pol N-terminal Domain Table 1. Structural statistics Restraints All NOE distance restraints Intraresidue Interresidue sequential (ji ÿ jj ˆ 1) Interresidue medium range (1 < ji ÿ jj 4 5) Interresidue long range (ji ÿ jj > 5) Hydrogen bonds Dihedral angles Acceptance criteria Ê NOE violations above 0.3 A Dihedral angle violations over 3  Remaining violations Ê NOE violations above 0.2 A Dihedral angle violations over 2  Ê NOE violations above 0.1 A Dihedral angle violations over 1  RMSDs from idealized covalent geometry Ê) Bonds (A Bond angles (deg.) Improper torsions (deg.) RMSDs from experimental constraints Ê) Distances (A Dihedral angles (deg.) Final energies (kcal/mol) Overall Bonds Angles van der Waals Improper torsions Distance restraints Dihedral angle restraints

Refined

Original

921 110

533 35

255

145

363

212

193 36 135 {SA} 0

141 22 68 hSAir 0

0

0

0.12(0.33)

0

0.16(0.37) 6.56(1.08)

0 6

1.72(0.84)

1

0.003(0.000) 0.357( 0.011) 0.261(0.010)

0.003 0.369 0.252

0.018(0.001) 0.207(0.031)

0.011 0.174

124.5(5.85) 12.48(0.54) 49.88(3.09) 44.38(1.72) 7.15(1.71) 10.30(7.18) 0.37(0.12)

129.3 13.52 53.41 46.27 6.69 9.13 0.26

{SA}, the 25 structures calculated with DYANA and energy minimized in X-PLOR 3.851 with force constants as described previously (Liu et al., 1996). hSAi, the mean of the {SA} structures and hSAir, the restrained minimization of the hSAi structure.

did not show this correspondence. The precision of the structure was primarily improved by collection of data at 600 MHz using gradient methods for water suppression, thereby allowing NOE data to be obtained to exchanging amide protons within the loop segments. The structure of the b-Pol N-terminal domain is formed from two pairs of antiparallel helices with the pairs packing with a crossing angle of approximately 60  , as previously reported (Liu et al., 1996). When comparing the minimized means between the re®ned and original structures the termini of helices 2-4 are either the same or vary by a single residue. However, helix-1 is extended from F25 to N28. The extension of helix-1 by almost a full helical turn causes major differences in the backbone and side-chain placements throughout the C terminus of helix-1 and the -loop. When the structures are aligned using the four helices the average difference between Ca distances in the

Ê . In addition to E26-A32 region are 6.6(1.1) A large backbone differences, the side-chain orientations for residues E26-N28 and A32 are rotated by nearly 180  with respect to the original structure. These changes in the backbone and sidechains also cause signi®cant alterations in solvent accessibility with residues E26 and A32 becoming less exposed and residues V29-S30 becoming substantially more exposed. Relaxation parameters The relaxation of the 15N nucleus have been interpreted in terms of the motion of the 1H-15N bond vector arising from the overall tumbling of the protein and from local motions of the bond vector. The R1 and R2 relaxation rates along with the 1H-15N NOE values are dependent on the amplitude of the spectral density function at ®ve frequencies: J(0), J(oN), J(oH ÿ oN), J(oH), and J(oH ‡ oN) (Abragam, 1961). The form of the spectral density function that was derived on the basis of the 1H-15N bond vector as a rigid rotor has been presented previously (Jardetzky & Roberts, 1981). Three experimental parameters, R1, R2, and 1H-15N NOE were determined at 11.75 T and 14.1 T. While measurement of additional relaxation rates can in principle enable a calculation of J(o) at all ®ve frequencies (Peng & Wagner, 1992a), for understanding the motions which lead to relaxation, this is not necessary. 15N relaxation data (R1 and R2 and 1 H-15N NOE values), were obtained for 76 of 86 residues at 500 and 600 MHz. Residues 7, 50 and 63 are proline residues and can not be observed, while residues 2-4, 12, 34, and 67-68 are missing due to chemical or conformational exchange. Figure 2 shows the R1 and R2 relaxation rates and the 1H-15N NOE values along with their estimated errors, and Table 3 shows the average rates for various secondary structural elements. All values listed in the text will come from the 500 MHz data unless otherwise noted. Nitrogen R1 rates At 500 MHz 15N R1 relaxation rates ranged from 2.47 to 1.28 sÿ1 with an average of 2.12 sÿ1. The average uncertainty at both 500 and 600 MHz is 5.9 %. The majority of residues have an R1 rate near the ``core'' average of 2.18 sÿ1 except for those near the N and C termini, where they are reduced (Figure 2(a)). As would be expected, rates at 500 MHz were faster then at 600 MHz by approximately 1.2 times. Nitrogen R2 rates 15

N R2 rates vary considerably from 19.21 to 1.83 sÿ1 with an average of 9.64 sÿ1 and have an average uncertainty of 6.8 % at both 500 and 600 MHz (Figure 2(b)). If the N and C termini are excluded the average R2 rate is 10.24 sÿ1. Residues near the N- and C termini have reduced transverse

232

Backbone Dynamics of the -Pol N-terminal Domain

Figure 1. NMR solution structure of the N-terminal domain of DNA polymerase b. (a) An overlay of 25 superimposed conformers (residues 11-80) showing several of the side-chains important for hydrophobic packing, DNA binding and catalysis. The structure is composed of four helices (15-28, 35-47, 56-61 and 68-78). The backbone of residues 11-55 are shown in magenta while residues 56-80 are in green. Hydrophobic side-chains are colored gold, hydrophilic brown, negatively charged red, and positively charged blue. (b) A backbone trace of 25 superimposed conformers of residues 10-80 demonstrating the results from the 15N relaxation measurements. The color code is as follows: blue, residues ®t with S2 only; gold, residues with slow conformational exchange (Rex); red, residues with slow internal motions (te); green, residues ®t with both Rex and te. (c) A ribbon representation for the minimized average structures (residues 9-82) illustrating residues with signi®cant chemical shift changes upon addition of DNA. Colors are from a consensus of chemical shift effects for four DNA fragments; a 5mer ssDNA, a p(dT8) ssDNA, a 9mer ssDNA containing a single U at the center, and a 12mer dsDNA fragment. No signi®cant changes in the surface for DNA binding occurs between the four DNA fragments. Colors represent the degree of chemical shift perturbations as follows: red > gold > green > blue.

relaxation rates with an average R2 of 7.01 sÿ1. A total of nine residues (T10, L11, N28, S30, Q31, I33, N37, I53, and K61) had R2 rates greater than 11.94 sÿ1 (one standard deviation above the mean core value). Residues N12, H34, and T67-K68 are missing in the 1H-15N correlated spectra due to exchange broadening. The -loop and helix-2 with mean R2 rates of 12.19 and 10.83 sÿ1, respectively, are increased with respect to other regions. Heteronuclear NOE values 1

H-15N NOE values ranged from 0.78 to ÿ1.12 with an average of 0.69 for all residues excluding the N and C termini (Figure 2(c)). The average uncertainty for NOE values is 16 % at both 500 and 600 MHz. Distinct differences were observed for the various secondary structural elements, with the helices having slightly larger mean NOE values (0.70) than the turns (0.65). Of the four helices, the helix-1/helix-2 (15-28, 35-47) antiparallel pair appear to be more rigid with an average NOE value of 0.72 compared to 0.68 for the helix-3/ helix-4 (56-61, 68-78) antiparallel pair. Turn 2 (4855) appears to be the most ¯exible with an average NOE of 0.62 compared to 0.67 for the -loop (29-

34) and the segment (62-67) that includes the hairpin turn. The N and C-terminal segments (2-14, 8087) show a large variation in NOE values ranging from ÿ1.12 for K5 up to 0.56 for I11 with an average NOE of ÿ0.12 indicative of sub-nanosecond motion. R2/R1 ratios The R2/R1 ratio for residues not undergoing large amplitude motions or exchange were used to estimate an initial tm value. The average ratio obtained at 500 and 600 MHz was 4.58(0.07) and 5.94(0.08), respectively, which correspond to tm values of 7.23(0.08) and 7.12(0.06) ns. The initial tm for the model selection was ®xed at 7.18 ns. This value is consistent with the correlation time expected for an 87 residue monomeric protein as determined from a correlation of tm versus molecular size for a number of proteins studied by 15N relaxation (Figure 3(a)). R2/R1 ratios were also used to test if an axially symmetric tumbling model provides a better ®t to the experimental data than an isotropic model. Using the method described by Tjandra et al. (1995), an initial diffusion tensor (Dk/D?) of 1.18 was determined. Using

233

Backbone Dynamics of the -Pol N-terminal Domain Table 2. Pairwise RMSDs Region RMSD of {SA} structures versus hSAi Folded chain (13-80) Helices (15-28, 35-47, 56-61, 68-78)) Helix-1 (15-28)) Helix-2 (35-47)) Helix-3 (56-61)) Helix-4 (68-78))

-loop (29-34)) Turn-2 (48-55)) Hairpin turn (62-67))

Refined

Original

Backbone

Heavy

Backbone

Heavy

0.37(0.05) 0.27(0.06) 0.24(0.04) 0.28(0.05) 0.36(0.06) 0.25(0.05) 0.32(0.05) 0.41(0.08) 0.49(0.10)

0.78(0.04) 0.58(0.34) 0.54(0.27) 0.54(0.33) 0.64(0.33) 0.64(0.45) 0.40(0.15) 0.91(0.51) 0.59(0.16)

1.09(0.21) 0.68(0.16) 0.68(0.22) 0.52(0.10) 0.78(0.16) 0.74(0.16) 1.63(0.73) 0.92(0.29) 1.16(0.21)

1.49(0.21) 1.15(0.17) 1.08(0.45) 0.85(0.40) 1.11(0.30) 1.12(0.35) 2.19(0.88) 1.49(0.49) 1.40(0.33)

{SA}, the ensemble of 25 structures; hSAi, the mean coordinates of the {SA} structures; hSAir, the restrained minimization of the hSAi structures.

the axially symmetric model with (Dk/D?) set to 1.18, only a marginal improvement in the w2 value was calculated in comparison to the w2 value obtained using the isotropic model. Application of

the F-test showed that this minor improvement was not statistically signi®cant. This is consistent with previous ®ndings that suggest that the Dk/D? typically needs to be greater than 1.5 for the

Figure 2. Relaxation rates for the free protein. Plots of (a) R1, (b) R2 and (c) 1H-15N NOEs and their uncertainties for the N-terminal domain at 500 and 600 MHz as a function of residue number and solution secondary structure. Data at 500 and 600 MHz are shown as ®lled and open circles, respectively.

234 Table 3.

Backbone Dynamics of the -Pol N-terminal Domain 15

N relaxation rates and 1H-15N NOEs at 500 and 600 MHz R1

Full ``Core'' Helices Helix-1 Helix-2 Helix-3 Helix-4

-loop Turn-2 Hairpin Termini

(500)

2.12(0.22) 2.18(0.12) 2.19(0.11) 2.15(0.77) 2.22(0.11) 2.24(0.17) 2.17(0.08) 2.15(0.12) 2.19(0.20) 2.08(0.07) 1.88(0.35)

R2

NOE(500)

(500)

9.64(2.82) 10.23(1.70) 10.10(1.24) 9.61(0.69) 10.83(1.54) 10.16(1.29) 9.68(0.92) 12.19(2.98) 10.47(1.81) 9.30(2.31) 7.01(4.81)

0.54(0.38) 0.69(0.05) 0.71(0.04) 0.72(0.03) 0.72(0.03) 0.67(0.04) 0.69(0.03) 0.67(0.06) 0.66(0.04) 0.62(0.03) ÿ0.12(0.52)

R1

(600)

1.74(0.17) 1.76(0.12) 1.77(0.10) 1.75(0.08) 1.78(0.10) 1.84(0.15) 1.73(0.08) 1.80(0.09) 1.77(0.21) 1.73(0.15) 1.58(0.27)

R2

(600)

10.13(3.05) 10.75(1.75) 10.67(1.26) 10.23(0.67) 11.39(1.56) 10.46(1.47) 10.35(0.95) 12.59(3.27) 10.90(1.91) 9.57(2.18) 7.40(5.44)

NOE(600) 0.62(0.35) 0.75(0.04) 0.76(0.03) 0.78(0.02) 0.78(0.02) 0.73(0.04) 0.74(0.02) 0.72(0.05) 0.74(0.04) 0.69(0.02) 0.04(0.51)

Values are the average rates and their standard deviations.

(R2/R1)max ÿ (R2/R1)min to exceed the uncertainty in the R2/R1 ratios by an order of magnitude (Tjandra et al., 1997). The reduced w2 value was calculated from the predicted and experimental R2/R1 ratios at various Dk/D? values using both the

NMR and X-ray structures (Figure 3(b)). The Dk/ D? value was found to be similar and near 1.0 in an analysis that used all structured residues (upper curves) versus an analysis that used all structured residues but excluded residues with large J(0)

Figure 3. (a) Rotational correlation time as a function of molecular size. Values are taken from 26 15 N relaxation studies (Akke et al., 1993b; Baldellon et al., 1998; Berglund et al., 1992; Cheng et al., 1993; Clore et al., 1990; Epstein et al., 1995; EvenaÈs et al., 1999; Gryk et al., 1998; Hodsdon & Cistola, 1997; Kay et al., 1996, 1989; KoÈrdel et al., 1992; Mandel et al., 1995, 1996; Markus et al., 1996; Peng & Wagner, 1992b; Powers et al., 1992; Red®eld et al., 1992; Sari et al., 1999; Schneider et al., 1992; Stivers et al., 1996; Stone et al., 1992, 1993; Yamasaki et al., 1995; Yu et al., 1996). All rotational correlation times were corrected to 25  C using the equation tm ˆ VhZ/kT, where V h ˆ hydrated volume, Z ˆ viscosity of water, k ˆ Boltzmann constant, and T ˆ temperature in Kelvin (Cantor & Schimmel, 1980). (b) Reduced w2 between experimentally determined R2/R1 ratios and the predicted R2/ R1 ratios at various Dk/D? ratios. The top two curves were determined using residues 13-79 and the bottom two curves used residues 13-79 but excluded residues with high J(0) values. The continuous lines represent the errors using the amide bond vectors of the mean NMR structure and the dotted lines represented the errors using the amide bond vectors of the crystal structure (Sawaya et al., 1997). The predicted R2/R1 ratios were determined at Dk/D? ratios from 0.1 to 2.6. The angle of the NH-bond vector for each residue was determined with respect to the long axis of the diffusion tensor calculated in the program MOLMOL (Koradi et al., 1996). The difference between the experimental and predicted R1 and R2 rates was minimized using the effective correlation time, where tm,eff ˆ (2Dk ‡ 4D?)ÿ1.

Backbone Dynamics of the -Pol N-terminal Domain

values (lower curves). The ®nding, that the reduced w2 value minimizes for the Dk/D? ratio of approximately 1.0 in all cases, suggests that the data is best ®t with an isotropic tumbling model. Model-free parameters (isotropic tumbling) The S2, te, Rex, and the ®tted model obtained from the model-free analysis assuming isotropic rotation are shown in Figure 4. For the N-terminal domain 37 residues were ®t with model 1 (S2); 16 residues were ®t with model 2 (S2 and te); 15 residues were ®t with model 3 (S2 and Rex); three residues were ®t with model 4 (S2, te and Rex); and ®ve residues were ®t with model 5 (S2s , S2f , and te). Of the 76 residues, only one, N87, was unable to be ®t adequately with any of the ®ve models; however, being the C-terminal residue and having an

235 NOE value of ÿ0.073 it is obvious that the residue is quite ¯exible. In ®tting the model-free parameters an optimized global tm value of 7.01(0.03) ns was determined. To be sure that there was good convergence at this value, the initial tm value was varied by  0.5 ns with no signi®cant difference in the ®nal optimized tm value. For residues 2-10 and 80-87 every residue had a te value included in the ®t with 14 of 24 residues that were ®t with a te term found at the N and C termini. Also, of the ®ve residues ®t using the extended model-free approach (model 5: S2s , S2f , and te) all were located in the termini. One may note that with S2f ˆ 1, model 5 reduces to model 2. In each case in which te exceeded 500 ps and model 5 was not used, model 2 was used, since this resulted in statistically more signi®cant ®ts than model 5 as evaluated from the F-test. Two exceptions to this

Figure 4. Model-free parameters for isotropic tumbling. Plots of the (a) order parameters (S2), (b) internal correlation times (te), and (c) Rex values as a function of residue number. Also shown are the solution secondary structure and uncertainties in the parameters for the model-free analysis which used a model of isotropic tumbling for the N-terminal domain. Errors in Rex are expressed as the average error using {1/[(1/s2i )]}0.5, and the Rex value is based on a two-site exchange model that uses 500 the equation R600 ex ˆ Rex (600/ 500)2 ˆ 1.44 R500 ex . The Rex values are reported with respect to 500 MHz. The Rex value at 500 MHz, weighted on the basis of its error, can be obtained using [(xi/s2i )]/ [(1/s2i )], where xi are the two Rex values determined by subtracting the R2 model-free value without Rex from the experimental R2 at 500 and 600 MHz and back correcting the Rex at 600 MHz using the equation, R500(calc) ˆ R600 ex ex /1.44. (d) The model-free model chosen for each residue (model 1, S2; model 2, S2 and te; model 3, S2 and Rex; model 4, S2, te and Rex and model 5, S2s , S2f and te).

236 were residues 55 and 61, which could not be ®t with either model 2 or model 5 but were ®t using model 4. An attempt at ®tting residues 55 and 61 to model 5 resulted in a S2f ˆ 1 (i.e. a poor model 2 ®t). In helix-1 (15-28) and helix-4 (68-78) all residues were ®t with a single S2 value except for A70 and F76 which had an Rex and te term added, respectively. For helix-2 (35-47) six of the 14 residues were ®t with an Rex term and for the -loop (29-34) four of seven residues were ®t with an Rex term. Thus, considerable conformational or chemical exchange processes occur in residues 27-48. H34 is absent in NH correlated spectra due to exchange processes. Helix-3 does not show any distinct pattern for model selection, however, a majority of residues in turn-2 (48-55) and the hairpin turn (61-67) ¯anking helix-3 were ®t with a te value. In addition to ps motion for the hairpin turn, chemical or conformational exchange appears to be present at the border between the hairpin turn and helix-4 based on T67 absence and K68 weakness in NH correlated spectra. In all helices, except helix-4, the residues near the C terminus were ®t with an Rex term. Regions of fast internal motions and conformational exchange have been mapped on the ribbon overlay of the structural conformers (Figure 1(b)). Model-free parameters (axially symmetric tumbling) For the N-terminal domain, an analysis of the atomic coordinates from the re®ned solution structure (residues 13-80) yields a axially symmetric molecule with an average inertia tensor of 1.00:1.08:1.88 calculated from the program MOLMOL (version 2.5.1) (Koradi et al., 1996). Even though the R2/R1 ratios demonstrated that the axially symmetric tumbling model was not statistically signi®cant, we included an analysis using this model in order to determine if the large Rex effects determined using the isotropic model would be in¯uenced by a model that utilized anisotropic tumbling. First, we considered the possibility that the Rex effects determined using the isotropic model could be an artifact of anisotropic rotation. Such anisotropic rotation would cause the spectral density function to be dependent upon the angle of the NH-bond vector with respect to the unique long axis of diffusion. The NH-bond vectors along helices 1 and 2 show relatively small angles with respect to the unique long axis of diffusion calculated for the folded domain (24(6)  for helix-1 and 36(12)  for helix-2), while residues in helices 3 and 4 show relatively large angles with respect to this axis (64(15)  for helix-3 and 57(15)  for helix-4) (Figure 5(a)). The experimental R2/R1 ratios as a function of their NH-bond angle and the experimental and predicted R2/R1 ratios for each of the core residues are shown in Figure 5(b) and (c). For a Dk/D? of 1.2, a value consistent with the structured portion of the domain, or for a less likely Dk/D? of 1.5, the predicted values of R2/R1

Backbone Dynamics of the -Pol N-terminal Domain

are no closer to the experimental values than are the predicted values of R2/R1 which use an isotropic tumbling model. As seen from Figure 5(b) and (c), the experimental and predicted R2/R1 ratios at either a Dk/D? of 1.2 (or alternatively at a Dk/D? of 1.5) do not show a correlation. In addition, many of the R2/R1 ratios are above 5.0, which is the highest predicted value possible assuming an axially symmetric molecule with a Dk/D? ˆ 1.5 and higher than the R2/R1 ratio predicted using a Dk/D? ˆ 1.2. These residues with high R2/R1 values are the same residues that display Rex. Clearly, the axially symmetric tumbling model can not predict these high values of R2/R1 within the error range of these values without the application of a Rex term. Among the largest R2/R1 values are found for residues in the -loop and helix-3, which are perpendicular to the long axis and should therefore show reduced values. Furthermore, R2/R1 values for helices-1 and 2 should be similar, because amide NH vectors in these antiparallel helices are each along the same long axis of the molecule. Contrary to this expectation, the data indicate increased R2/R1 values for several residues in helix-2 (Figure 5(c)). The model-free analysis that utilized a Dk/D? of 1.18 did not show a difference in those residues requiring a ®t with the Rex parameter. Furthermore, in a separate calculation, the optimized diffusion parameter (Dk/D?) was found to be 1.052, very similar to an isotropic molecule (Dk/D? ˆ 1). Reduced spectral density mapping The relaxation rates (R1, R2, and 1H-15N NOE) were used to determine the spectral density function at J(0), J(oN), and hJ(oH)i. Results are illustrated in Figure 6. Values of J(0) and J(oN) decrease while values of hJ(oH)i increase with greater internal motion. However, for cases of exchange, J(0) values are signi®cantly increased. All residues ®t with an Rex term in the model-free analysis had J(0) values above 3.0 ns/rad and only one residue, S30, had a J(0) value above 3.0 and was not ®t with Rex, J(oN) was relatively insensitive to minor changes in ¯exibility, while hJ(oH)i was sensitive to minor changes in ¯exibility. Reduced spectral density mapping for the ssDNA complex The model-free analysis method was not applicable to the ssDNA complex due to exchange of the b-Pol N-terminal domain both with and along the ssDNA. Furthermore, intensity loss suggested that for a fraction of the ssDNA, more than one protein domain was associated with the ssDNA, producing severely broadened resonances that did not contribute to the observed resonance intensity. The relaxation rates for the observable resonances of the b-Pol N-terminal domain-ssDNA complex were amenable to spectral density mapping, and the values of J(0), J(oN) and hJ(oH)i were deter-

Backbone Dynamics of the -Pol N-terminal Domain

237

Figure 5. (a) Amide NH-bondvector angles relative to the long axis of the molecule and their standard deviations for the 25 NMR solution structures. The N and Cterminal tails were not used in the calculation of the diffusion tensors. (b) The R2/R1 ratios versus amide NH-bond-vector angle with respect to the long axis of the molecule. Solid circles represent the experimentally determined R2/R1 ratios, the solid curve shows the predicted R2/R1 ratios that were calculated using a Dk/D? of 1.2 and the dashed curve shows the predicted R2/R1 ratios that were calculated using a Dk/D? of 1.5. (c) Experimental and predicted R2/R1 ratios at 500 MHz for residues 13-79. Filled triangles are the experimentally determined values, solid circles show predicted values using a Dk/D? of 1.2, open circles show predicted values using a Dk/D? of 1.5, and the continuous line assumes isotropic tumbling. Predicted R2/R1 ratios were calculated holding Dk/D? equal to 1.2 or 1.5 as described in Figure 3(b).

mined for comparison to the free protein. Relaxation rates acquired at 11.75 T for an N-terminal/ 5mer-ssDNA complex were used to determine the spectral density functions at J(0), J(oN) and J(oH). In addition to crosspeaks for residues that were absent for the uncomplexed N-terminal domain, crosspeaks for K5, F25, A47, A70 and I73 showed spectral overlap, and the crosspeak for I69 was missing due to exchange broadening. Differences in the spectral density functions between the uncomplexed and protein/5mer-ssDNA complex are shown in Figure 7. Values of J(0) in the complex were reduced for a majority of residues by approximately 0.2 ns/rad. Signi®cant increases in J(0) were observed for L11, S30, G64 and G66 due to increases in measured R2 rates. Residues L11 and S30 are very weak in HSQC spectra and are therefore very dif®cult to accurately measure. However, the increased J(0) for G64 and G66 are well within the error of the measurements. In addition to the changes observed for G64 and G66, we note that I69 shifts dramatically down®eld in the 1H dimension on addition of the 5mer ssDNA in the initial titration and disappears due to a much increased R2 for either 1H or 15N at the end

of the titration. Residues G64, G66 and I69 show three of the largest shifts upon binding 5mer ssDNA (Figure 8(a)). The increased exchange at G64, G66, and I69 is likely due to sliding on the DNA strand or the binding and releasing of the DNA molecule. The spectral density function at J(oN) on average is increased by approximately 0.04 ns/rad as compared to the free protein. The increase in J(oN) and reduction in J(0) is consistent with the complex tumbling at a faster rate then the uncomplexed protein and was not expected. This is almost certainly due to the lower concentration of protein in the 5mer-ssDNA complex. Values at hJ(oH)i showed no signi®cant changes upon binding 5mer-ssDNA complex. All dynamics that is further presented refers to the b-Pol N-terminal domain alone except where speci®ed. Dynamics analysis: N and C-terminal segments The N (2-12) and C (80-87) termini of the Nterminal domain showed an average S2 of 0.50 indicative of a large degree of high frequency motion. Reduced spectral density mapping showed reduced values for J(0), J(oN) and marked increases

238

Backbone Dynamics of the -Pol N-terminal Domain

Figure 6. Reduced spectral density mapping for the free protein. Plots of (a) J(0), (b) J(oN) and (c) Jh(oH)i and their uncertainties as determined from reduced spectral density mapping at 500 and 600 MHz as a function of residue number and solution secondary structure. Data at 500 and 600 MHz are shown as ®lled and open circles, respectively.

in hJ(oH)i. The increase in hJ(oH)i for these residues is similarly indicative of high frequency motion. These regions are unstructured in the re®ned solution structure. The Rex was absent in the termini except for T10 and L11 which show large Rex values of 8.3 and 7.4 sÿ1 and large J(0) values of 4.7 and 5.6 ns/rad, respectively. N12 shows exchange contributions based on the absence of a cross peak in NH correlated spectra. G13 and G14 connect the highly ¯exible N terminus to helix-1 and are well ordered. Dynamics analysis: helices The four helices have an average S2 value of 0.94. The helix-1/helix-2 antiparallel pair with an average S2 of 0.96 and average hJ(oH)i of 0.0094 rad/second showed more restricted motion than the helix-3/helix-4 antiparallel pair with an S2 value of 0.92 and hJ(oH)i of 0.0111 rad/second. Helices 3 and 4 displayed slightly higher RMSD in

the re®ned solution structure as well (Table 2). Helix-1 and helix-4 show almost no Rex contributions. However, both helix-2 and helix-3 show considerable exchange effects with six of 13 residues in helix-2, and two of ®ve residues in helix-3, being ®t with an Rex term. For each of these residues, that were ®t with an Rex term, the J(0) value was increased. Dynamics analysis: loops and turns The average S2 for turns is 0.88 and the average hJ(oH)i is 0.012 showing that they are more ¯exible than the helices with an S2 of 0.94 and hJ(oH)i of 0.010. The hairpin turn (61-67) has the lowest S2 and largest hJ(oH)i consistent with it displaying the most internal motion of the three turns. The

-loop (29-34) has an average S2 value of 0.93, similar to the values for helices. However, this value appears to be greater than would be expected based on the relaxation rates and the

Backbone Dynamics of the -Pol N-terminal Domain

239

Figure 7. Reduced spectral density mapping for the 5mer complex. Plots of the changes in (a) J(0), (b) J(oN) and (c) J(oH) upon the binding of a 5-mer ssDNA (50 -GCTAT30 ) and their uncertainties at 500 MHz as a function of residue number and solution secondary structure. Differences were obtained by subtracting the values of the free protein from the complex.

spectral density at hJ(oH)i. Residues in the -loop and turn-2 (48-55) have considerable Rex contributions with three of six residues in the -loop and four of seven residues in turn-2 having Rex values between 0.6 sÿ1 and 6.2 sÿ1. Each of the residues ®t with an Rex term also had increased values of J(0). One exception was S30 which has an increased J(0) value of 3.81 rad/seconds consistent with exchange, but was not ®t with an Rex term in the model-free analysis. This is due to the large errors for the R1, R2 and 1H-15N NOE for S30 allowing the simple S2 model to adequately ®t the data. Oligonucleotide binding Three single-stranded oligomers of various lengths were chosen for binding studies as possible analogues of a single-stranded region of a gapped DNA substrate, while a 12mer duplex was chosen for binding studies in order to investigate possible duplex-DNA interactions with the b-Pol N-terminal domain. The U nucleotide in the 9mer can

be converted into an abasic site substrate for the AP lyase of the b-Pol N-terminal domain by uracil DNA glycosylase. The three singlestranded oligonucleotides 50 -GTCAT-30 (5mer), 50 -CTGCUGATG-30 (9U), p(dT)8, and the doublestranded oligionucleotide 50 -CACCACGTGGTG-30 (dsDNA-12mer) have been shown to induce large 1 H and 15N amide proton chemical shift changes at the HhH motif, at the -loop, and at the adjacent helix-2. The 5mer, 9U and dsDNA-12mer were part of this study, while the effects of p(dT)8 have been reported previously (Liu et al., 1996). Figure 8 show the 1H and 15N chemical shift changes for the various DNA-protein complexes. Signi®cant changes of 50.05 ppm for 1H and 50.4 ppm for 15 N are localized in C-terminal residues in the

-loop, the N-terminal half of helix-2, helix-3, the b-turn and helix-4 regions. Helix-1 and the connecting segment from 48-55 show the least effect upon binding. On binding the 5mer and 9U oligonucleotides, each of the HhH amide protons, except for K61, V65, E71, and T79 display down-

240

Backbone Dynamics of the -Pol N-terminal Domain

Figure 8. (Legend opposite)

®eld chemical shift changes. An identical pattern was observed for the p(dT)8 oligonucleotide with the exception of A70 and A78 which were shifted up®eld and T79 which was shifted down®eld. The results suggest that the b-Pol N-terminal domain contacts dsDNA and ssDNA at a similar interaction surface. Those residues showing consensus chemical shift effects on binding the four DNA oligonucleotides are shown in the ribbon representation of the re®ned solution structure (Figure 1(c)).

Discussion Models for isotropic versus axially symmetric tumbling The core of the N-terminal domain (residues 1379) forms a prolate ellipsoid, and thus care should

be taken in order to ensure that the Rex effects are not a manifestation of anisotropic tumbling. As shown in Figure 5(b), no correlation is observed between measured R2/R1 ratios and the angular dependence of the amide-bond vector with respect to the long axis of the protein domain. Furthermore, no type of dimerization can be envisioned that would allow NH vectors in helix-2 of the antiparallel helix-1/helix-2 pair to show high anisotropy without helix-1 similarly experiencing the anisotropy (i.e. high R2/R1 values are observed for several amides in helix-2, while consistently low R2/R1 values are observed for amides in helix-1). While the structure of the ordered residues suggest that an anisotropic tumbling model would be appropriate, the relaxation data experimentally ®ts the isotropic tumbling model. While dimerization

241

Backbone Dynamics of the -Pol N-terminal Domain

Figure 8. Chemical shift changes upon DNA binding. Plots of the changes in backbone-amide proton and nitrogen chemical shifts of the N-terminal domain upon binding (a), (e) 5mer ssDNA (50 -GCTAT-30 ) (b), (f) p(dT)8 (c), (g) 9mer ssDNA (50 -CTGCUGATG-30 ) and (d), (h) a 12mer dsDNA (50 -CACCACGTGGTG-30 ) as a function of residue number and solution secondary structure. Changes were calculated by subtracting the values of the free protein from the complex.

could in principle produce a more spherical molecule, a plot of tm versus molecular size for correlation times determined by 15N relaxation (Figure 3(a)) and previous sedimentation equilibrium results indicate that the b-Pol N-terminal domain behaves as a monomer (Dimitriadis et al., 1998). A likely explanation for the isotropic relaxation behavior is found on examination of the approximate positions of the N and C-terminal tails with respect to the folded structure (Figure 9). Bax and co-workers demonstrated that removing three ¯exible residues of ubiquiton reduces the effective rotational correlation time by 0.21 ns, based on hydrodynamic calculations, suggesting that ¯exible tails in¯uence tm signi®cantly (Tjandra et al., 1995). While the N and C termini of the Nterminal domain are quite ¯exible they will have a large radius of gyration and have a large amount

of bound water, which will likely cause them to have a signi®cant effect on the rotational correlation time. Figure 9 shows that the N and C-terminal tails always act to increase the dimensions of the two perpendicular axis without affecting the parallel axis signi®cantly. Thus, it is likely that the large ¯exible tails will act to slow rotation about the D? axis causing the molecule to act isotropically or at least within the error of measurement of the anisotropy. Backbone dynamics at the HhH motif Of signi®cant interest is the manner by which backbone motion relates to DNA binding (Figure 1(c)) or dRP lyase catalysis (Scheme 1) for the b-Pol N-terminal domain. Estimated rate constants of the backbone motion for residues that

242

Backbone Dynamics of the -Pol N-terminal Domain

HN

HN

H NH 2

H3N

O

O3PO

O P

H3N

O

Lys-35

O

O

O

H

HR OH

HS

O

H H H N H

H H

Lys-68 O

Tyr-39

His-34

H

O

Lys-68

N

N O3PO

His-34

P

H

O

O

H

Lys-72

H3N

O

Lys-35

O

Tyr-39 Lys-72

H O3PO

H

HR OH H H

O

P

OH

H

H

Lys-72 O Lys-35

O

Tyr-39

O

H

H3N

O

HR HS

H

H N H

H

O

O3PO

O

HS O

N

O H O H P O O

H H3N Lys-35

His-34

HN

Tyr-39 N O3PO OH

H

H

OH

H

H

P

H

Lys-72 O

O

H

H

OH

O

H

H

N

H

O

O3PO

O

H3N Lys-35

Scheme 1.

contribute to catalysis or directly contact DNA are shown in Table 4. Backbone motion on slow and fast time scales was present at the HhH motif in the b-Pol N-terminal domain (Figure 4). Backbone motion on a slow (millisecond) time-scale was estimated for two lysine residues (K68 and K60) that participate in DNA binding (Table 4). The slow Rex motion for K60 and exchange at K68 appear to be due to a more general conformational exchange effect observed at the termini of helices (see below). Residues L62 and V65 in the hairpin turn, that participate in DNA binding, show motions on a fast time-scale. For the L62-P63-G64-V65 hairpin, the re®ned solution structure reveals a weak Ê , 9.9-22  ) between the hydrogen bond (2.64-2.68 A 2 amide proton of V65 (S ˆ 0.869) and the carbonyl oxygen atom at L62 (Figure 10(a)). This exact sequence of ®ve residues is conserved in the HhH motifs of at least eight unique DNA repair enzymes and proteins (E. coli, Bacillus subtilis, and Caenorhabditis elegans endonuclease III, E. coli and

Saccharomyces cerevisiae MutY, Micrococcus luteus UV endonuclease, Thermus aquatus and Thermus scoductus DNA ligase, Saccharomyces cerevisiae Rev1 (a repair polymerase), E. coli RecR (Doherty et al., 1996; Mullen & Wilson, 1997), and in each case where structures are determined the turn is exposed like in the b-Pol N-terminal domain for DNA interaction and the Leu residue packs against the remainder of the structure. In the b-Pol singlenucleotide gap complex, the L62 carbonyl oxygen atom coordinates to the Na‡, presumably weakening the effect of V65 hydrogen bonding via the same sp2 orbital, and may similarly coordinate Na‡ in solution. Na‡ coordination is completed by the carbonyl oxygen atoms of K60 and V65, a phosphate group on the DNA and water. As judged from the reduced spectral density mapping results, the single-stranded 5mer-oligonucleotide complex showed no signi®cant changes in the high frequency backbone motion at the HhH on binding a single-stranded oligonucleotide. However, this is a

Backbone Dynamics of the -Pol N-terminal Domain

243

Figure 9. Overlay of the N and C-terminal tails of the 25 NMR solution structures along the three principal diffusion axes. The diffusion axes were determined for residues 13-79 and are annotated with Dk for the long axis and D?a and D?b for the two short axis. The ribbon representation of the core residues (13-79) is for the mean of the 25 NMR structures. The N-terminal tails are shown in blue and the Cterminal tails are shown in green.

weakly binding oligomer, and motional restriction on binding gapped or incised abasic site dsDNA can not be ruled out. Potential restriction of internal motion for the L62-G66 segment on binding a DNA substrate could produce a maximal unfavorable entropic contribution of 1.2 kcal molÿ1

to binding as calculated using equation 7 by Akke et al. (1993a) by assuming full restriction on binding DNA. It is unlikely that this energy cost (1.2 kcal molÿ1) would reduce DNA binding af®nity, but instead when balanced by the enhanced contacts that would result from adaptation of the

244

Backbone Dynamics of the -Pol N-terminal Domain

Table 4. Backbone motion for residues contributing to DNA binding or dRP lyase activity in the b-Pol N-terminal (8 kDa) domain Residue

L62 V65 K72 K35 Y39 H34 K60 K68

S2

a

0.873 0.869 0.990 1.000 0.967 ex 0.998 ex

Values or limits for 1/te and kbex

Functional role dRP Lyase activityc

ssDNA bindingd

gapped dsDNA bindinge

dRP binding pocketf

ND ND ‡ ‡ ‡ ‡ ‡ ÿ

ND ND ÿ ‡ ND ‡ ‡ ‡

‡ (bb)j ‡ (bb)j ‡ (sc) ‡ (sc) ÿ ‡ (sc) ‡ (bb)j ‡ (bb)

ÿ ÿ ‡ ‡ ‡ ÿ ÿ ÿ

1/te ˆ (9.8(0.4))  108 sÿ1 1/te > 1  1010 sÿ1 g 1/te > 1  1010 sÿ1 g rigid rotor 1/te > 1  1010 sÿ1 g kex < 1  102 sÿ1 h kex < 5  104 sÿ1 kex < 5  104 sÿ1 i

a

Model free order parameter from an isotropic ®t. The kex limit was estimated on the basis of the Rex equation below assuming a 50:50 population of two states with a chemical shift difference of 450 Hz (Mandel et al., 1996):    kex tcp p1 p2 …
o†2 2 Rex ˆ 1ÿ tanh kex tcp kex 2 b

c Reduction in dRPase activity was found in b-Pol 8 kDa mutants with native helical structure and with residual activity as follows: K72A/K68A/K35A < K72A  K72A/K68A < K60A  H34G  K35A < E75A < F25W < E71Q  K68A/E71Q  K84A  K68A  wild-type (Prasad et al., 1998). K72Q, K72R, and Y39Q were assessed as having reduced dRP lyase activity (Matsumoto et al., 1998). ND indicates that the activity was not determined for a mutation. d ssDNA binding activity was assessed for several mutants described in footnote c (Prasad et al., 1998). The K72Q and K72R mutants had damaged DNA binding activity exceeding wild-type b-Pol 8 kDa domain (Matsumoto et al., 1998). e DNA contacts made by residues in the single-nucleotide gap DNA complex of b-Pol (Sawaya et al., 1997), where sc denotes a side-chain contact and bb denotes a backbone contact. f The residues forming the dRP binding pocket were assessed from the structure and the effects of residue mutations on dRP lyase activity. Two water molecules are located within this pocket in the single nucleotide gap DNA complex with b-Pol. g For model 1, limits on te are 10 ps. h This residue is not observed in the 1H-15N HSQC and kex line broadening of the 1H or 15N resonance in excess of the JNH coupling constant (100 sÿ1) would broaden this peak beyond observation. i The kex for K68 for which resonance intensity is lost in the ®rst R2 time point is at least as fast as for K60. j The backbone contacts consist of carbonyl coordination of a Na‡ coordinated at a phosphodiester oxygen atom.

backbone, would be expected to increase DNA binding. Assuming a simple model of diffusion in a cone by the NH-bond vector, substantial cone semi-angles for internal motion are calculated to be 17  , 24  , 17  and 18  for L62, G64, V65 and G66, respectively. In this model, the 15N nucleus is positioned at the tip of the cone, and a semi-angle of 0  corresponds to no internal motion (S2 ˆ 1), while a semi-angle of 90  corresponds to complete internal motion (S2 ˆ 0). Within helix-3 of the HhH, semi-angles for internal motion are found to be 22  , 20  , 12  and 14  for residues G56, A57, E58 and A59, respectively. The high frequency motion in these backbone amides as well as those in the hairpin turn are among the highest for residues that form regular secondary structure within the structural core. The G56 amide proton has a down®eld chemical shift of 10.04 ppm that is characteristic of a strong hydrogen bond to the side-chain carboxylate of D74, as determined from the re®ned structure. Interestingly, this hydrogen bonding does not restrict the internal motion of the G56 NH bond. K60, a DNA binding residue as determined by sitedirected mutagenesis, has nearly fully restricted high frequency backbone motion (S2 ˆ 0.998). An Rex contribution at K60 suggests slow backbone motion at this residue (discussed below).

Rex effects at the termini of helices All residues that show Rex contributions or severely broadened resonances are well-structured in the re®ned structure except for the segment T10L11-N12, where very large Rex effects were observed (Figure 1(b)). Although not obvious from the structure, weak NOE values between protons in the 10-12 segment and the 52-54 segment suggests a transient association between these regions of the polypeptide backbone, possibly through b-strand pairing. Such pairing would explain the down®eld amide proton shifts for K52 and K54 and could be an important, although transient, stabilizing feature in the structure. Slow time scale Rex effects result from an amide 15 N nucleus experiencing an exchange between two (or more) states with altered 15N chemical shifts. In general, chemical shifts are highly sensitive to small electronic effects such as hydration and hydrogen bonding, proximity of charge, or changes in electron density. Making and breaking of amide-hydrogen bonds on a slow time scale could contribute to chemical shift differences in 15 N between two states for amides at or near the C termini of helix-1 (K27, N28), helix-2 (I46, A47, K48, Y49), and helix-3 (A59, K60, K61). Examination of the group of 25 re®ned conformers shows

Backbone Dynamics of the -Pol N-terminal Domain

245

Figure 10. The hairpin turn of bPol and the helix-hairpin-helix (HhH) motifs of b-Pol, AlkA and endonuclease III. (a) An overlay of 25 superimposed structural conformers (residues 61-66) illustrating the hydrogen bond between the amide proton of V65 and the carbonyl oxygen atom of L62. The distance between the amide proton and carbonyl oxygen atom ranges Ê . (b) A superimfrom 2.64 to 2.68 A position of the HhH motifs of the re®ned N-terminal domain of DNA polymerase b (residues 59-76), AlkA (residues 209-226) and endonuclease III (residues 111-128). The labeled side-chains K60, K68 and K72 are for DNA polymerase b and correspond to Q210, W218, and Y222 in AlkA and E112, K120, and V124 in endonuclease III.

hydrogen-bonded versus non-hydrogen-bonded amides at residue 28 at the C terminus of helix-1, residues 47 and 48 at the C terminus of helix-2, and residue 61 at the C terminus of helix-3. Exposed amides at the N-termini of the helices would be affected by water solvation. Different 15N (and 1H) chemical shift environments are possible for different water solvation states at residues at or near the N terminus of helix-1 (T10, L11), helix-2 (I33, H34), helix-3 (G56), and helix-4 (T67, K68). The Rex contributions to amide groups at both the N and C termini of the helices are consistent with local helix-coil transitions on a slow time-scale. For the ssDNA oligonucleotide complex, T10-L11 retained the large exchange contributions to the zero frequency spectral density J(0). Similarly, exchange contributions within other segments were unchanged within the error of the measurements. For T67, conformational exchange was slowed in a p(dT)8 complex, thereby allowing the direct assignment of the 1H and 15N resonances for this amide (Liu et al., 1996). Binding of various DNA oligomersconformational effects The chemical shift effects on binding both ssDNA and dsDNA oligomers of different lengths

indicate similar interaction mechanisms. The HhH apparently interacts with ssDNA and dsDNA similarly. The -loop/helix-2 appears to show versatility in its interactions with different residues being affected by ssDNA of various lengths. Binding to the dsDNA 12mer likely occurs via interaction by the -loop at the end of the duplex in a manner similar to that in the gapped DNA complex (Sawaya et al., 1997). Independent measurements of the Ca chemical shifts suggest slight increases in backbone helicity as measured by the CSI at the N and C termini of helix-1 on binding a ssDNA 5mer. The effects on helix-1 are apparently propagated from DNA contacts at the -loop and at the C terminus of helix-1. No large effects on 1H or 15N shifts for the amides were observed in helix-1. On binding p(dT)8, the JNHa for K61 at the C terminus of helix-3 was found to change from 7.3 Hz to 4.9 Hz, indicating a change toward helicity for this residue (Liu et al., 1996). On binding each of three ssDNA oligonucleotides as well as a dsDNA oligomer, large down®eld amide proton chemical shift changes were observed for several of the amide proton resonances within the HhH. Down®eld shifted amide protons participate in relatively strong hydrogen bonds as compared to those that are weakly hydrogen bonded (Tjandra & Bax, 1997). Since not all amide protons in the HhH

246 helices are affected by phosphate contacts or proximity, it is reasonable to assume that amide protons that experience a down®eld shift, that are not in proximity to the DNA, have stronger hydrogen bonds in the complex. The V65 amide-proton resonance in the HhH hairpin shifts up®eld for all cases of ssDNA and dsDNA binding examined. Similarly, the E71 amide-proton resonance in helix4 characteristically shifts up®eld in each case. Weakening of V65 hydrogen-bonding is likely in the complex due to Na‡ binding to the L62 carbonyl oxygen atom, which is the hydrogen-bond acceptor for the V65 amide proton. The source of the up®eld shift for E71 is not obvious, but amide hydrogen bond weakening to the T67 carbonyl oxygen atom is possible. An E71Q mutation had no affect on ssDNA binding ruling out possible carboxylate coordination of a Na‡ in a DNA complex. Relating the refined solution structure and dynamics to the high-resolution gapped DNA-b b-Pol crystal structure The precision of the presently determined solution structure and the high-resolution and goodquality electron density seen for the b-Pol N-terminal domain in complex with gapped DNA (Sawaya et al., 1997) allow a comparison of these structures. Differences in these structures would appear to result from gapped DNA binding. The helix-hairpin-helix is highly similar in the two structures, although there is slight unwinding at the C terminus of helix-4 in solution. The most pronounced differences in these structures appears to

Backbone Dynamics of the -Pol N-terminal Domain

result form the gapped DNA interaction by the

-loop and helix-2 with the DNA in the crystal structure. The overlay of the two structures shows conformational differences in the -loop, in the position of helix-2, and in the C-terminal turn of helix-1 (Figure 11). In solution helix-1 shows a slight kink, and helix-2 has a slight curvature. The overall effect, results in a slight left-handed supercoiling of helix-1/helix-2, and this effect is not seen in the X-ray structure. The conformational differences at the C-terminal end of helix-1, in the -loop, and in helix-2 correlate with Rex contributions within these segments. Conformational exchange within these segments of the structure may therefore facilitate the conformational change on binding the gapped DNA. The backbone changes in these segments dramatically affect the side-chain positions of H34 and K35. The H34 side-chain in the solution structure is not positioned for template base contact as seen in the crystal structure due to large differences in both the backbone and in the w1 torsion angle (‡37(1.9)  in solution as compared to ÿ60  in the gapped DNA complex). Thus, helix-2 would appear to undergo a conformation change that rotates the helix away from the core of the domain and toward the DNA gap. This motion could facilitate an initial interaction of the -loop and helix-2 with the incised abasic site. H34 might be expected to interact initially with the abasic-site hole, since the

-loop can not penetrate into a incised abasic site DNA duplex as seen in the crystal structure in the absence of the large 90  DNA bend.

Figure 11. Comparison of re®ned NMR solution structure of b-Pol to the gapped DNA b-Pol crystal structure. Ribbon overlay for residues 13-80 of a representative structure (#1) and the gapped DNA crystal structure of DNA polymerase b. The ribbon for the NMR solution structure is shown in red while the crystal structure is shown in green. Side-chains for residues H34, K35, Y39, K60, K68 and K72 which have been shown to be important for DNA binding and/or catalysis are shown in magenta for the NMR structure and light green in the crystal structure. Gapped DNA from the crystal structure is shown in blue.

247

Backbone Dynamics of the -Pol N-terminal Domain

The role of backbone motion in dRP lyase catalysis

High frequency motion at the N and C-terminal tails

Restricted motion is seen at the dRP-lyase activesite pocket formed by residues K72, Y39, and K35. The dRP active-site pocket is shallow, and primarily accommodates the aldehyde carbonyl group and the phosphate group to be eliminated. K72 directly participates in Schiff's base formation on the ring opened form of the 50 -dRP group bound at the active-site pocket. In the previously proposed mechanism (Scheme 1), Y39 promotes proton transfer to the C1 aldehydic oxygen of the dRP group, and at the same time provides a mechanism for deprotonation of the attacking K72 nucleophile. K35 was previously suggested to participate in binding and/or stabilization of the leaving phosphate group, and a K35A mutant shows a 50 % reduction in dRP lyase activity under non-saturating substrate conditions. K35 contributes to catalysis in the K72A single mutant, as judged by a 10 % residual activity that is lost in a K68A/K72A/ K35A triple mutant but not in a K72A/K68A double mutant (Prasad et al., 1998). The rigidity of the backbone at K72, Y39, and K35 allows a wellformed active-site region. Restricted backbone motion at active site residues is in contrast to the backbone motion observed for active-site residues in the 4-oxalocrotonate tautomerase and the ®nding that the backbone motion at the active-site residues was restricted on binding substrate (Stivers et al., 1996).

High frequency backbone motion in the C-terminal tail can be attributed to this being outside the folded domain. The C-terminal tail is not connected to the 31 kDa polymerase domain, and therefore provides information on the intrinsic ¯exibility of this sequence, which does not pack with the N-terminal domain or 31 kDa domain as determined by X-ray crystallography. The high frequency motion within the N-terminal ten residues of the b-Pol N-terminal domain is signi®cant in view of the high conservation of this unstructured segment across species. Perhaps, the ¯exible K3-R4-K5-A6-P7 segment is important in nuclear localization of b-Pol or for an interaction that is yet to be determined. The N-terminal segment would be an optimal nuclear localization sequence without the A6 residue insert.

Exchange effects in the -loop/helix-2relationship to dRPase catalysis A slow-time scale Rex effect has been observed for Q31 and I33 in this study. The adjacent H34 resonance is broadened beyond observation. These results are characteristic of conformational exchange within the -loop and the N-terminal end of helix-2. The imidazole ring of H34 provides a stacking contact within the DNA at the template base in the single nucleotide gap complex (Sawaya et al., 1997) and may interact initially in the abasic site hole at incised abasic sites (Mullen & Wilson, 1997). Additionally, the side-chain of H34 has a reduced pKa, and has been postulated to be involved in opening of the hemiacetal through deprotonation at O10 (Mullen et al., 1997). A H34G mutation reduced dRP lyase activity by 50 % (Prasad et al., 1998). The rate constant for dRP catalysis is 0.1 sÿ1 and is considerably slower than the rate constant for conformational change at H34, which can be estimated to be on the order of 100 sÿ1. Thus, this slow conformational exchange for H34 will not contribute to the rate of catalysis, at least not for the substrate used in the previous study. To be slow enough to affect catalysis, the different conformations at H34 would have resulted in distinct resonances for the two conformational states.

Comparison of HhH structures An overlay of the HhH structures for the re®ned solution structure of the b-Pol N-terminal domain, E. coli endonuclease III, and AlkA is shown in Figure 10(b). In each case the HhH motifs do not have a cation bound. The RMSDs for backbone atoms for a superimposition of the HhH in the b-Pol N-terminal domain (residues 59-76) determined here and the HhH in AlkA (residues 209226) and endonuclease III (residues 111-128) are Ê and 1.10 A Ê , respectively. This compares to 1.17 A Ê on superimposing the same an RMSD of 0.80 A regions of the re®ned NMR solution structure and the X-ray structure of b-Pol in the single-nucleotide gap with Na‡ bound. The sequence LPGVG is highly conserved in HhH motifs, and potential motion in the LPGVG hairpin, which we have described here, has been previously considered on the basis of high B-factors in the structure of endonuclease III (Kuo et al., 1992). The high-frequency motion in the LPGVG hairpin may be a feature found in all HhH motifs with this sequence. Such motion could allow for slight adaptations of the HhH for productive DNA contacts.

Materials and Methods Sample preparation The N-terminal domain of rat DNA polymerase b (residues 2-87) was overproduced in the E. coli strain BL21/pLysS/pRSET-8k (Dr Steve Widen, University of Texas Medical Branch) grown on minimal media containing 15NH4Cl as the sole nitrogen source. Overproduction of the N-terminal domain and the puri®cation procedure has been described (Liu et al., 1994; Prasad et al., 1993). In preparing the NMR samples, the puri®ed N-terminal domain was applied to a Sephadex G15 column (18 cm  1.0 cm) and eluted with 5 mM Tris-d11 (pH 6.7), 100 mM NaCl at 5  C. All buffers were passed through Chelex-100 resin before use. The ®nal protein concentration for relaxation experiments was approximately 2 mM. The 1H and 15N resonance

248 assignments were used as described (Liu et al., 1994), except for Asn12 which was reassigned as Asn87 based on HNCA and HNCOCA triple resonance experiments. Two single-stranded DNA oligonucleotides, a 5mer (50 -GCTAT-30 ) and 9mer (50 -CTGCUGATG-30 ) and a double-stranded 12mer (50 -CACCACGTGGTG-30 ) were synthesized on a MilliGen/Biosearch Cyclone Plus DNA synthesizer. The DNA, with an attached DMT group, was puri®ed by HPLC using a BioRad C-18 reversephase column using a gradient from 5 % to 65 % acetonitrile with 100 mM NaOAc (pH 6.5). After puri®cation the DMT protecting group was removed by treatment with 80 % acetic acid for 20 minutes at room temperature. After neutralizing with NaOH the DNA fragments were puri®ed by reverse phase HPLC using a gradient from 0 % to 100 % acetonitrile over 30 minutes. DNA samples were lyophilized to a powder, dissolved in water to 30 mM and had the pH adjusted to 6.8. Each of these DNA samples was titrated directly into the NMR sample. Final sample conditions were 1.0 mM protein, 3.2 mM DNA, 100 mM NaCl, 5 mM Tris-d11 (pH 6.8) for the protein-5mer complex, 1.2 mM protein, 3.9 mM DNA, 400 mM NaCl, 5 mM Tris-d11 (pH 6.8) for the protein-9mer complex, and 0.3 mM protein, 0.3 mM DNA, 1 M NaCl, 5 mM Tris-d11 (pH 6.8) for the proteindsDNA complex. NMR measurements All NMR experiments were recorded at 25  C on Varian Unity-Plus 500 and 600 MHz spectrometers each equipped with a triple-resonance pulse ®eld Z-axis gradient probe and gradient ampli®er. Two channels were used with channel 1 equipped with a waveform generator. All experiments were acquired employing experiments with sensitivity enhancement and gradients to select for coherence transfer pathways and each used States-TPPI for quadrature detection in the indirect dimensions (Marion et al., 1989). Sweep widths of 13.3 ppm for 1H, centered on the water resonance, 27.5 ppm for 15N, centered at 119 ppm, and 30.0 ppm for 13C, centered at 54 ppm were used for all experiments. The 3D 1 H-15N-edited nuclear Overhauser enhancement spectroscopy: heteronuclear single quantum coherence (NOESY-HSQC) spectra were acquired at 600 MHz with 512 (t3)  128 (t1)  32 (t2) complex points using a mixing time of 150 ms and 16 scans per increment. The 1H-15N HSQC spectra for DNA titrations were acquired at 600 MHz with 512 (t2)  128 (t1) complex points with 16 scans per increment. HNCA and HN(CO)CA experiments were acquired with constant time evolution at 600 MHz with 512 (t3)  80 (t1)  32 (t2) complex points with 16 scans per increment. The relaxation experiments (15N R1, R2 and 1H-15N NOE) were as described by Farrow et al. (1994). Proton decoupling used a series of 550 ms 180  pulses, that were applied during the relaxation period of the R1 experiments to eliminate the contributions of dipolar cross-correlation and chemical shift anisotropy to the longitudinal relaxation (Kay et al., 1992). For R2 experiments, a 90  15 N pulse followed by a gradient pulse was applied at the beginning of the sequence to ensure that magnetization originated on 1H and not 15N (Farrow et al., 1994). A 15 N CPMG sequence (Carr & Purcell, 1954; Meiboom & Gill, 1958) with 80 ms 180  pulses spaced 0.9 ms apart was used in the R2 experiments to minimize resonance offset effects and ®eld inhomogeneity. Cross-correlation

Backbone Dynamics of the -Pol N-terminal Domain effects on 15N relaxation were eliminated through use of 180  proton pulses applied with every second echo in the CPMG sequence. Due to the excellent water suppression achieved by use of gradients and a hard ¯ip-back pulse (2030 ms) at the water resonance (Farrow et al., 1994), no additional solvent suppression or time domain deconvolution techniques were needed. This eliminated the possible contribution of proton exchange NOEs from water. A total of 1024 (t2)  128 (t1) complex points were recorded with four scans per increment for both R1 and R2 experiments and 32 scans per increment for 1H-15N NOE experiments. 15N decoupling during the acquisition was achieved using a WALTZ decoupling scheme (Shaka et al., 1983). For R1 measurements ten delays of 11, (67), 133, (266), 389, (555), 770, (1121), 1754, and 2498 ms and for R2 measurements ten delays of 16, (32), 48, (64), 80, (96), 127, (175), (239), and 319 ms were used with duplicates in parenthesis. For both R1 and R2 experiments, a recycle delay of 1.5 seconds was used. Heteronuclear 1H-15N NOE experiments were recorded in the presence and absence of a proton saturation period of three seconds during the recycle delay which was ®ve seconds. The 1H saturation was achieved with the use of 120  1H pulses applied every 5 ms (Markley et al., 1971). Two sets of experiments were recorded and the values of the NOE values averaged. All spectra were processed and analyzed using the software Felix95 (Biosym Inc.) on Silicon Graphics R4400 computers. For R1, R2, 1H-15N NOE, and HSQC experiments the ®nal data sets consisted of 2048 (t2)  1024 (t1) real data points after zero-®lling and Fourier transformation. Linear prediction was used to extend the t1 dimension from 128 to 256 complex points. For 3D spectra the ®nal data sets consisted of 1024 (t3)  512 (t1)  128 (t2) real data points, and linear prediction was used to extend the 15N dimensions from 32 to 64 complex points and the 13C dimensions from 80 to 160 complex points. Apodization was performed using squared sine bell window functions typically with a shift of 60  for all spectra. Solution structure refinement The coordinates for the 25 re®ned conformers (1DK2) and the minimized mean structure (1DK3) have been deposited in the Protein Data Bank. The solution structure of the N-terminal domain of DNA polymerase b has been re®ned employing a complete analysis of a 15N-edited 3D NOESY data set collected using gradient water suppression techniques at 600 MHz. This data set was supplemented with the previous 13C-edited NOESY data, homonuclear NOESY data, and JNHa and Jab coupling constant data obtained from 2D HMQC-J and DQF-COSY spectra. Crosspeaks were quanti®ed by measuring volumes and classi®ed as strong, medium, or weak with upper Ê , 4.0 A Ê and 5.0 A Ê , respectively. distances limits of 2.5 A Calibrations of NOE distance restraints were as described in detail previously (Liu et al., 1996). The number of useful upper bound distance restraints was 921 as compared to 533 for the original structure calculation (Liu et al., 1996). In addition, 67 torsion angle restraints were added based on the 13Ca chemical shifts. The structural conformers were determined from the distance and angle restraints using torsion angle dynamics in DYANA (version 1.5) (GuÈntert et al., 1997). The calculation started with 100 randomized

249

Backbone Dynamics of the -Pol N-terminal Domain conformers and was annealed using 7000 steps. The 50 structures with the lowest target function were re®ned using simulated annealing in X-PLOR (version 3.851) by heating to 1000 K followed by 500 cooling steps (BruÈnger, 1992). No structures had NOE violaÊ and or torsion angle violations greater than 0.3 A tions greater than 3  . The 25 lowest energy conformers displaying the least number of Ramachandran violations were selected to represent the NMR ensemble. Within residues 13-80, the 25 structures showed 87 % f, c torsion angles within most favorable regions, 13 % in additionally allowed regions and none in generously or disallowed regions according to the program DYANA (version 1.5).

R1 ˆ

R2 ˆ

  1 3 2 d ‡ c2 ‰4J…0† ‡ 3J…oN †Š 6 4

and R2 ‰4J…0† ‡ 3J…oN †Š ˆ 6J…oN † R1 as given by others (Fushman et al., 1994; Gryk et al., 1998). Substituting the spectral density function described here into the R2/R1 ratio yields: R2 7 ‡ 4…oN tm †2 ˆ R1 6

Relaxation parameter calculations All crosspeak heights in the R1, R2, and 1H-15N NOE spectra were measured using the program Felix 95 (Biosym). Uncertainties in the measured peak heights were extracted from the root-mean-square baseline noise of the spectra. Differences between the values obtained from duplicate spectra were within these errors. Longitudinal relaxation rates, R1, were calculated from the equation I(t) ˆ I1 ÿ [I1 ÿ I0]exp(ÿR1t). Transverse relaxation rates, R2, were calculated from the equation I(t) ˆ I0 exp(ÿR2t). In each case, the 15N R1 and R2 relaxation rates for each residue were determined using a non-linear least squares ®t of the experimental data. Fits were performed using the Levenburg-Marquardt algorithm (Press et al., 1986) from modi®ed scripts provided with the model-free (version 3.1) software. As a check, ®ts were performed using the program SigmaPlot (Jandel Scienti®c). There were no differences in the rates that were determined using the two programs. Uncertainties in R1 and R2 relaxation rates were estimated by performing 500 Monte Carlo simulations based on the estimated errors in the measured peak heights (Kamath & Shriver, 1989; Palmer et al., 1991). Steady state 1H-15N NOE values were determined from the ratio of the average peak heights in spectra recorded with proton saturation to those recorded without saturation. The uncertainties for the NOE values were calculated using the relative errors of the peaks.

  1 3 2 d ‡ c2 ‰3J…oN †Š 3 4

Residues with internal motion on a time-scale longer than several hundred picoseconds (i.e. those having an NOE value <0.60) were excluded from the tm calculation, as were residues with conformational or chemical exchange, as identi®ed by having R2 rates greater than one standard deviation above the mean R2 rate for residues with NOE values >0.60. Model-free approach assuming isotropic tumbling The relaxation data was used to calculate model-free parameters for ®ve different models as described (model 1, S2; model 2, S2 and te; model 3, S2 and Rex; model 4, S2, te, and Rex; and model 5, S2, Sf, and te) (Mandel et al., 1995; Stone et al., 1992). Initially all ®ve models were ®t to the experimental data while holding the overall rotational tumbling time (tm) constant at 7.1 ns. A fullgrid search of all optimized parameters was performed with S2s and S2f varied from 0 to 1.0, in steps of 0.02; te varied from 0 to 3000 ps, in steps of ®ve ps; and Rex varied from 0 to 15 sÿ1, in steps of 0.25 sÿ1. The w2 between the predicted and the experimental data was minimized for each of the models in the model-free program. The w2 was compared for each of the models and the statistical signi®cance of a reduction in w2 was checked with the F-test. 500 Monte Carlo simulations were performed to generate simulated errors in the parameters for each model.

R2/R1 ratios: isotropic tumbling

Model selection

In order to analyze the relaxation data using the model-free formalism an initial global rotational correlation time was ®rst determined. The 15N R2/R1 ratio is, to a good approximation, independent of rapid internal motions and of the magnitude of the chemical shift anisotropy (Kay et al., 1989). For residues that have large order parameters (S2 > 0.6) and fast internal motions (te < 100 ps) the spectral density function is approximated by:

In all cases the simplest model that adequately satis®ed the data was chosen. In the selection, the w2 value, as described above, was compared with the 95 % con®dence interval of the w2 distribution obtained from the Monte Carlo simulations in the model-free (version 4.0) program. The choice of a Rex model (3 or 4) was con®rmed by a J(0) value that exceeded 3.0 ns/rad in all cases except for residue 30. A model was chosen if the w2 value was less than the critical value. A more complex model was chosen if the following criteria were met: (1) the w2 of the simpler model was above the critical value; (2) the w2 of the more complex model was below the critical value; and (3) the model could be statistically justi®ed in terms of the F-test statistic. The F-test statistic is F ˆ [v2(w21 ÿ w22)]/[(v1 ÿ v2)w22] where w21 and w22 are the values for models with v1 and v2 degrees of freedom (v1 > v2). In order to be statistically signi®cant the F-test statistic comparing the two models must be greater than the critical value of the F-distribution determined at the

J…o† ˆ

  2 S2 tm 5 1 ‡ …otm †2

which is the simple rigid rotor model incorporating S2. If the overall correlation time for the protein is slow (tm > 3 ns) then the 15N R1 and R2 relaxation rates can be approximated by assuming that the spectral density values at high frequencies J(oH ‡ oN), J(oH), and J(oH ÿ oN) do not contribute yielding:

250

Backbone Dynamics of the -Pol N-terminal Domain

95 % con®dence interval based on synthetic data sets that were obtained from 500 Monte Carlo simulations. This procedure was repeated for every added parameter. Once the models were selected for each individual residue the model-free analysis was repeated using the correct model for each residue. In this case the global rotational correlation time was optimized after a fullgrid search from 6.5 to 7.5 ns in steps of 0.05 ns. Uncertainties in the ®nal optimized parameters were calculated by carrying out 500 Monte Carlo simulations (Palmer et al., 1991).

R2/R1 ratios-axially symmetric tumbling For axially symmetric tumbling, an initial diffusion coef®cient D, where D ˆ Dk/D?, was estimated. This was performed using the method described by Tjandra et al. (1995) which minimizes the difference between the observed and predicted R2/R1 ratios: " #2    X R2  R2 w ˆ ÿ s2obs;n R1 calc;n R1 obs;n n

Reduced spectral density mapping The relaxation data was analyzed by reduced spectral density mapping using software generously provided by Wagner and co-workers (Farrow et al., 1995; Ishima & Nagayama, 1995a,b; Lefevere et al., 1996). Spectral densities for individual residues were calculated at J(0), J(oN) and hJ(oH)i at both 500 and 600 MHz. Uncertainties were determined from 500 Monte Carlo simulations (Kamath & Shriver, 1989). DNA titrations Single stranded DNA fragments were titrated directly into the NMR samples containing the b-Pol N-terminal domain, and HSQC spectra were acquired at each titration point, thereby allowing each crosspeak to be tracked upon addition of DNA. Chemical shift differences were determined by subtracting the free chemical shift from the bound chemical shift. Assignments for the 9mer-protein complex were con®rmed using HNCA and HN(CO)CA experiments.

2

where s is the estimated error in the experimentally measured R2/R1 ratio, obs is the residue speci®c experimentally measured ratio, and calc is the calculated ratio based on the rate equations as given in the Supplementary Material. The summation extends over n residues excluding those having large amplitude internal motions or exchange contributions as described earlier. No te was used in the calculation of an initial diffusion coef®cient. This procedure was repeated twice once with one variable, holding Dxx ˆ Dyy ˆ Dzz (isotropic rotation) and again with two variables Dxx and Dyy by holding Dyy ˆ Dzz (axially symmetric rotation). An F-test (Devore, 1982; Ratkowsky, 1983) was used to assess whether there was a statistically signi®cant improvement of ®t for the axially symmetric tumbling model over the isotropic model.

Model-free approach assuming axially symmetric tumbling The relaxation data were also interpreted in terms of a model assuming an axially symmetric tumbling. The general approach used in selecting the models was the same as that for the isotropic model. The steps were as follows: The initial value for the global rotational correlation time was kept at 7.18 ns, and the initial value for the diffusion constant was set to 1.18 as described above. In order to minimize the calculation time no Monte Carlo simulations were performed in the initial model selection. For a majority of residues it was obvious from the w2 values alone as to which model was appropriate. For the other residues the model-free calculation was repeated using 500 Monte Carlo simulations and the F-test was performed. Dk/D? was ®xed at 1.18 during the model selection process and during the entire modelfree calculation. A separate model-free calculation was performed that used the appropriate model for each residue and optimized both tm and Dk/D? after a complete grid search from 6.5 to 7.5 ns, in steps of 0.05 ns for tm and from 1.0 to 1.7, in steps of 0.05 for Dk/D?. The ®nal optimized values were 7.01 ns for tm and 1.052 for Dk/D?. Uncertainties were calculated as for the isotropic model.

Acknowledgements This research was supported by grant GM52738 from the National Institutes of Health (G.P.M.) and a postdoctoral fellowship grant GM18956 from the National Institutes of Health (M.W.M). We thank Dr L.E. Kay for the availability of pulse sequences for 15N relaxation and 1 H-15N NOE measurements. We also thank Dr A. Palmer III for the availability of the model-free program for analysis and Dr G. Wagner for the reduced spectral density mapping software. We thank Martha Santos for synthesis and puri®cation of the ssDNA oligonucleotides, and Dr Linda Olson for synthesis of the 12mer duplex DNA. We thank Dr Walfrido Antuch for collection of the NMR data on the 12mer-DNA complex.

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Edited by P. E. Wright

(Received 7 July 1999; received in revised form 8 December 1999; accepted 9 December 1999)

http://www.academicpress.com/jmb Supplementary material is available from JMB Online