Human DNA Polymerase Î¼ Can Uses a Noncanonical Mechanism for

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Human DNA Polymerase µ Can Uses a Noncanonical Mechanism for Multiple Mn -mediated Functions 2+

Yao-Kai Chang, Ya-Ping Huang, Xiao-Xia Liu, Tzu-Ping Ko, Yoshitaka Bessho, Yoshiaki Kawano, Manuel Maestre-Reyna, Wen-Jin Wu, and Ming-Daw Tsai J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b01741 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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Journal of the American Chemical Society

Human DNA Polymerase  Can Use a Noncanonical Mechanism for Multiple Mn2+-mediated Functions

Yao-Kai Chang1,2, Ya-Ping Huang1, Xiao-Xia Liu1,#, Tzu-Ping Ko1, Yoshitaka Bessho1,3, Yoshiaki Kawano3, Manuel Maestre-Reyna1, Wen-Jin Wu1,*, and Ming-Daw Tsai1, 2,* 1Institute

of Biological Chemistry, Academia Sinica, 128 Academia Road Sec. 2,

Nankang, Taipei, 115, Taiwan, 2Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan. 3RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.

*Corresponding author: [email protected] or [email protected] #Present

address: Shanghai Jiao-Tong University School of Medicine, 280 South

Chongqing Road, Shanghai. P.R. China 200025

1 ACS Paragon Plus Environment

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Abstract Recent research on the structure and mechanism of DNA polymerases has continued to generate fundamentally important features, including a noncanonical pathway involving “prebinding” of metal-bound dNTP (MdNTP) in the absence of DNA. While this noncanonical mechanism was shown to be a possible subset for African swine fever DNA polymerase X (Pol X) and human Pol  it remains unknown whether it could be the primary pathway for a DNA polymerase. Pol  is a unique member of the X-family with multiple functions and with unusual Mn2+ preference. Here we report that Pol  not only prebinds MdNTP in a catalytically active conformation, but also exerts the Mn2+ over Mg2+ preference at this early stage of catalysis, for various functions – incorporation of dNTP into a single nucleotide gapped DNA, incorporation of rNTP in the non-homologous end joining (NHEJ) repair, incorporation of dNTP to a ssDNA, and incorporation of 8-oxo-dGTP opposite template dA (mismatched) or dC (matched). The structural basis of this noncanonical mechanism and Mn2+ over Mg2+ preference in these functions was analyzed by solving 19 structures of prebinding binary complexes, precatalytic ternary complexes, and product complexes. The results suggest that the noncanonical pathway is functionally relevant for the multiple functions of Pol . Overall, this work provides the structural and mechanistic basis for the long standing puzzle in the Mn2+ preference of Pol , and expands the landscape of the possible mechanisms of DNA polymerases to include both mechanistic pathways.


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INTRODUCTION

Despite half-century of research, new functions as well as mechanistic and structural features of DNA polymerases (pols) have continued to be uncovered, as reviewed recently.1-10 The issue of particular interest is the mechanistic and structural basis for the diverse functions and the broad spectrum of fidelity displayed by different DNA polymerases. Even for the enzymes within the same family, the X-family Pol , Pol  Pol  and African swine fever virus (AFSV) Pol X whose major roles are in DNA repair, the fidelity varies from 100,000 for Pol 11 to close to 2 for Pol X12. The mechanism of Pol  has been studied extensively and shown to follow the canonical mechanism of replicative pols, with DNA binding first followed by MgdNTP binding that triggers a subdomain-closing conformational change.2, 6, 13-15 On the other hand, Pol X, a half-sized viral pol lacking the 8 kDa/lyase subdomain and the duplex DNA binding subdomain, has been suggested to follow both the canonical mechanism for Watson-Crick base pair incorporation, and a noncanonical mechanism with MgdGTP binding before DNA (designated as “prebinding”) for the incorporation of a dG:dGTP (anti:syn) Hoogsteen base pair.16 This noncanonical mechanism was subsequently shown to play a role in modulating the fidelity of the medium-fidelity Pol .17 In bacteria, the DNA polymerase from Thermus thermophilus HB8 Pol X (tt-Pol X) also has the ability to bind MgdGTP before binding DNA.18 However, whether this noncanonical mechanism could be the primary reaction pathway of a DNA polymerase remains to be established. The X-family DNA polymerase  (Pol ) contains 494 a.a. with 24%, 31% and 3 ACS Paragon Plus Environment


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41% sequence identity to Pol , Pol  and terminal deoxynucleotidyltransferase (TdT), respectively.19-20 It contains a BRCT domain in addition to a Pol like DNA polymerase domain. Pol μ is the only known DNA polymerase that possesses both template-dependent activity and template-independent terminal transferase activity (TdT-like).21 Pol  plays a major role in non-homologous DNA end joining (NHEJ) repair.22-28 One unique role of Pol  in NHEJ is to align broken DNA ends that lack complementary sequences.28 The terminal transferase activity of Pol  has been proposed to be required to create or increase complementarity of DNA ends during NHEJ.27 Pol  also participates in light chain gene rearrangement of the immunoglobulin during V(D)J recombination.28-29 When transfected with equimolar ratio, Pol  competed moderately with TdT in N-nucleotide addition.29 Pol  has also been shown to display gap-filling activity for a single nucleotide (1-nt) gapped DNA substrate.22-23,

30

dNTP-stabilized

It also exhibits DNA slippage activity on reiterative DNA,31-32 misalignment

activity

on

non-reiterative

DNA,32-33

and

skipping-ahead activity in which Pol  skips the first available template base and incorporates a nucleotide to the template base at the 5′ end of the gap.24-25 Recently two tumor-associated Pol  mutations G174S and R175H located at the 8 kDa subdomain have been characterized.34 Almost all pols prefer Mg2+ over other divalent metal ions for their biological functions. Mn2+ ion is known to lower the fidelity of pols and is thus considered a mutagenic metal ion.35-44 In contrast to the stringent coordination requirement for Mg2+, Mn2+ has a more relaxed coordination requirement,43,

45

thus can tolerate

misalignment in catalyzing misincorporation.43 On the other hand, anti-oxidant 4 ACS Paragon Plus Environment

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enzyme such as human manganese superoxide dismutase requires Mn2+ for its function in protecting mitochondria against oxygen-mediated free radical damage.46 Mitochondria oxidative damage has also been linked to diabetes, aging and Parkinson's and Alzheimer's diseases.46 While Mn2+ ion is essential for the daily metabolic activity or detoxification in human body, too much of it can be toxic.47-49 Chronic manganese exposure may result in Mn accumulation and cause neuromotor and cognitive deficits.50-53 Pol  has a strong preference for Mn2+ over Mg2+ ions for its TdT-like activity21. In addition, Mn2+ ion increases the NHEJ efficiency of Pol , though with the cost of increased misincorporation.54 In the presence of Mn2+ ion, Pol  is highly error-prone in adding dNTP to a template-primer DNA.21 In the single nucleotide extension of a 3’-protruding dsDNA substrate, Pol  can add each of the four MnddNTP equally well using its terminal transferase activity.27 The molecular bases for these properties are still unknown. The first structure of Pol  in complex with a 1-nt gapped DNA and the incoming MgdTTP was reported by Moon et al.22 Uniquely, unlike Pol  and Pol  that undergo large conformational changes upon DNA binding,17,

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Pol 

undergoes minimal conformational changes, except loop 1, going from the apo form to DNA-bound, precatalytic ternary, and postcatalytic complexes, showing its rigidity during its catalytic reaction cycle.23 A recent time-lapse experiment has visualized the process of phosphodiester bond breaking and making during dNTP incorporation to a 1-nt gapped DNA,30 which shows that Pol  catalyzes the correct dTTP incorporation with a faster reaction rate using Mn2+ than Mg2+ at the chemical step. Interestingly, His329, a critical residue in positioning the primer terminus,22 interacts with the 5 ACS Paragon Plus Environment


-phosphate of dNTP or the pyrophosphate group only in the presence of Mn2+ but not Mg2+.30 In addition to dNTP, Pol  can also incorporate MgrNTP efficiently due to the lack of a steric gate residue (Tyr271 in Pol , Tyr505 in Pol  but Gly433 in Pol ).54, 56-58

By comparing the structures of precatalytic and postcatalytic complexes of Pol

 containing a ribonucleotide in the active site, Moon et al. showed that Pol  can accommodate and incorporate an rNTP similarly to a dNTP without distortion in the active site geometry.59 Like Mn2+, rNTP also enhances NHEJ by Pol .54 Recently, Pryor et al. showed that as much as 65% of cellular NHEJ products contain transiently embedded ribonucleotides, and rNTP but not dNTP incorporation by Pol  (or TdT) promotes the ligation of broken chromosomes during NHEJ.26 In another direction, 8-oxo-dGTP is a highly abundant dGTP oxidation product accounting for up to 10% of the dGTP pool.60 Due to its dual coding potential, 8-oxo-dGTP is mutagenic once incorporated into a genome as it can either form an anti:syn dA:8-oxo-dGTP mispair or an anti:anti dC:8-oxo-dGTP correct base pair,61 as has been well demonstrated for both Pol 62 and Pol 63. 8-oxo-dGTP incorporation has also been reported to occur for Pol X64. Very recently, Çağlayan and Wilson showed that Pol  can insert 8-oxo-dGTP opposite a template dT even more efficiently than opposite a template dA or dC, and handed off the repair intermediate to DNA ligase I to seal the nick.65 In this work we showed that the functionally relevant Mn2+-nucleotide complexes, but not the corresponding Mg2+-nucleotide complexes, prebind Pol  with high affinity in the absence of DNA. Furthermore, analysis of 19 crystal structures of 6 ACS Paragon Plus Environment

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various Pol  complexes indicated that Pol  effectively orients various Mn2+-nucleotide complexes to catalytically ready conformations at the prebinding stage. The results suggest that the noncanonical, prebinding mechanism is a functionally relevant mechanism for Pol 

MATERIALS AND METHODS This section describes only key experimental methods. Additional materials and procedures are described in Supporting Information (SI) Materials and Methods.

Protein Expression and Puriﬁcation. For crystallization purpose, the human Pol μ loop 2 deletion mutant containing the polymerase core with residues 132-494 (referred as "hPol μ Δ2" in the original publication by Moon et al.,23 for which the residues between Pro398-Pro410 were replaced with a glycine residue) was cloned into the pGEXM vector and expressed in BL21CodonPlus (DE3)-RIPL competent cells (Agilent Technologies) at 16 °C for overnight. The proteins were purified following the published procedure23 with slight modification. In brief, GST-Pol μ Δ2 with a TEV-protease cleavage site was purified via glutathione agarose (Thermo Scientific), and the GST-tag was cleaved by on-resin TEV cleavage at 25 °C for 1 hour. Subsequently, Pol μ Δ2 was purified by size-exclusion chromatography using a Superdex 75 16/60 (GE Healthcare) with a buffer containing 25 mM Tris, 80 mM NaCl, 5% glycerol, and 1 mM DTT at pH 8. Finally, the purified Pol μ was concentrated to 16.4 mg/mL and was flash-frozen in liquid nitrogen for crystallization 7 ACS Paragon Plus Environment


and for storage. For ITC measurements, Pol  (a.a. 128-494) without loop 2 deletion was prepared. This construct was cloned into pET15b vector with an N-terminal His-tag and a TEV-protease cleavage site. The expression and purification procedure was the same as that for GST-Pol μ Δ2 except for the first affinity column purification step, which was done via cOmplete™ His-tag purification resin (Sigma-Aldrich) and eluted with the lysis buffer containing 200 mM imidazole. The His-tag was cleaved in solution by TEV protease at 25 °C for 1 hour, then the proteins were further purified by size-exclusion chromatography.

Data Collection and Structural Determination. The crystals were placed onto a stream of nitrogen gas at 100 K. The X-ray diffraction data were collected at a wavelength of 1.0 Å using the beamlines of TPS 05A (Rayonix MX300-HX CCD detector) and BL15A1 (Rayonix MX300-HE CCD detector) at the National Synchrotron Radiation Research Center (NSRRC) located in Hsinchu Taiwan, and the beamline of BL32XU (Rayonix MX225-HE CCD detector) at Super Photon ring-8 GeV (SPring-8) in Japan. The data were processed by using the software HKL200066 or XDS67. All structures were determined by molecular replacement using Phaser-MR68 in CCP4 suite69. Apo Pol  (PDB code: 4LZD23) was used as the starting model for solving the binary complex structures of Pol :MndNTP, Pol :MgdNTP, Pol :MnrNTP, and Pol :Mn8-oxo-dGTP. The published Pol  ternary complex coordinate (PDB code: 4M0423) was used as the starting model for solving the structure of all ternary complexes. The further refinement was performed by Refmac569 in CCP4 suite and iteratively built in COOT.70-71 8 ACS Paragon Plus Environment

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RESULTS Outline and Experimental Sets. As shown in Table 1, we performed three sets of studies for Mg2+ versus Mn2+ specificity: 1-nt gap-filling by dNTP, incorporation of rNTP, and 8-oxo-dGTP incorporation opposite template dA and template dC. In all three sets, we determined the Kd value of metal-nucleotide binding to Pol  and solved their structures. For sets 1 and 3, we further solved the structures of the corresponding precatalytic complexes and product complexes if they are not available from previous reports. Then we compared the structures of the data and structures at each stage of the reaction, between Mg2+ and Mn2+ complexes.

Table 1. Summary of experimental sets and 19 structures from this study, and previously reported structures used for comparison. Nucleotide

Metal

E:M·Nucleotide

DNA

binary dNTP

Mn2+

Structure 1-4

Mg2+

Structure 5-7

Precatalytic

Product

ternary

complex

(ATGC)

dUMPNPP

rNTP

Mn2+

1-nt gap_dAa

Structure 8

Jamsen et al.30

Mg2+

1-nt gap_dA

Moon et al.23

Jamsen et al.30

Mn2+

Structure 9-12

(AUGC) UMPNPP

Mg2+

1-nt gap_dA

Moon et al.59

8-oxo-dGTP

Ca2+

1-nt gap_dA

Structure 14

Mn2+

a1-nt

Structure 13

1-nt gap_dA

Structure 16

Mg2+

1-nt gap_dA

Structure 17

Ca2+

1-nt gap_dCb

Mn2+

1-nt gap_dC

Structure 18

Mg2+

1-nt gap_dC

Structure 19

Structure 15

gap DNA with template dA. Template:5′-CGGCATACG-3′, the upstream primer 9 ACS Paragon Plus Environment


5′-CGTA-3′, and the 5′-phosphorylated downstream primer 5′-pGCCG-3′. b1-nt gap DNA

with template dC. Template:5′-CGGCCATACG-3′, the upstream primer 5′-CGTA-3′, and the 5′-phosphorylated downstream primer 5′-pGCCG-3′.

Apo Pol  Binds Mn2+-nucleotide Tightly but Mg2+-nucleotide Weakly. Previously we reported that, in the absence of DNA, both Pol X16 and Pol  show high MgdNTP affinity in the low micro-molar Kd range. For Pol , which has the highest fidelity in the X family, the binding of MgdNTP in the absence of DNA was too weak to be measured by ITC.16 Like Pol , Pol  also has a very low MgdNTP affinity with Kd values in the range of 140-230 M as (Table 2 and Figure S1). However, surprisingly, Pol μ binds MndNTPs tightly, with Kd values in the range of 0.22-1.95 M, suggesting that Pol μ not only can prebind MdNTP, but also exerts its functional metal ion specificity at the prebinding step. For the prebinding of MndNTP to be functionally relevant, it is necessary to show that Pol μ does not bind DNA much more tightly than MndNTP. Thus we also measured the Kd values for two types of DNA that are relevant to the two different functions of Pol μ: ssDNA for the TdT-like template-independent DNA synthesis, and dsDNA with a 1-nt gap for gap-filling (Figure S1). As shown in the last row of Table 2, the affinity of Pol  to a 12-mer poly dT ssDNA (Kd = 11.05±1.31 M) is weaker than MndNTPs (Kd = 0.22-1.95 M), while that of a 1-nt gapped DNA (Kd = 1.90±0.30 M) is closer to but still weaker than MndNTPs. Since the physiological concentrations of dNTPs should be higher than those of DNAs, the results taken together suggest that the prebinding of dNTP should be a feasible, and likely the preferred pathway under physiological conditions. 10 ACS Paragon Plus Environment

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Table 2. The Kd values (M) for the binding of dNTP, rNTP, 8-oxo-dGTP to Pol a in the absence of DNA determined by ITCb; for comparison, the measured Kd values (M) for DNA substrates are also included. row

metal

Ligand Kd (M)

1

Mn2+

dGTP 0.95±0.30c dGTP 140±4 rGTP 0.75±0.16 rGTP 98±2 ssDNA (12-mer dT) 11.05±1.31

dATP dCTP dTTP 8-oxo-dGTP 0.22±0.02 1.95±0.42 1.62±0.14 6.7±1.2 2 Mg2+ dATP dCTP dTTP 8-oxo-dGTP 153±6 206±16 230±10 191±8 3 Mn2+ rATP rCTP rUTP 0.71±0.21 2.76±0.47 1.77±0.60 4 Mg2+ rATP rCTP rUTP 115±9 236±46 168±12 5 Mn2+ 1-nt gapped DNAd 1.90±0.30 aWild type Pol  without the loop 2 deletion. bAll samples contain 50 mM borate/NaOH, 200 mM KCl and 4 mM MnCl2 or 10 mM MgCl2 at pH 7.0. cThe ± value stands for standard deviation from three repeats.

dTemplate:5′-CGGCATACG-3′,

the upstream primer

5′-CGTA-3′, and the 5′-phosphorylated downstream primer 5′-pGCCG-3′.

Structural Basis for the MndNTP over MgdNTP Preference of Apo Pol μ. To understand the molecular basis of the high affinity of MndNTP to Pol , we solved the structures of Pol μ:MndNTP binary complexes with all four dNTPs (Structures 1-4 in Table 1, with structural statistics shown in Tables S1). We used the Pol  polymerase domain with its loop 2 (Pro398-Pro410) replaced with a glycine residue (see Methods), which has been shown not to affect Pol μ's functions in DNA gap-filling, NHEJ reaction, and template-independent DNA synthesis.23 As shown with an example in Figure 1A, there is no significant global conformational change in the protein upon MndCTP binding (r.m.s. deviation of 0.218 Å over 288 aligned C atoms). However, there are notable conformational changes in the active site (Figure 11 ACS Paragon Plus Environment


1B, omit map of MndCTP shown in Figure S2): the carboxylate groups of Asp328, Asp330 and Asp418 rotate to chelate the two manganese ions, and the side chains of Arg323 and Lys325 interact with the  and  phosphates of dCTP, respectively. The rotation of Asp330 side chain (2.1 Å displacement for the 2 oxygen) upon MndCTP binding vacates the space to accommodate His329 side chain. His329 side chain flips drastically to have its N1 atom form a plausible hydrogen bond with -phosphate of dCTP, and Trp434 side chain rotates substantially to make room to accommodate the ribose portion of dCTP. Similar structures were also observed for the other three Pol μ:MndNTP binary complexes (Figure S3), except that, unlike the previously reported Pol :MgdNTP binary complexes for which the conformations of the nucleotide bases of the four dNTPs are more converged due to the pi-pi stacking interactions with the side chain of Tyr505, the base moieties of the four different dNTPs in Pol  appear to be capable of sampling different conformations, likely due to the small Gly433 residue (instead of the corresponding bulky Tyr505 in Pol ) which results in the lack of pi-pi stacking interactions. Another contributing factor is the lack of base pairing since DNA is not yet bound at this Pol :MndNTP binary complex stage. Remarkably, the results show that in the absence of DNA, both the nucleotide binding metal Mn2+ B and the catalytic metal Mn2+ A are already present in the Pol :MndNTP binary complexes and in the Pol :MnrNTP binary complexes (described in a later section) as confirmed by the anomalous difference density maps (Figure S4), suggesting that Pol  can be preactivated by MndNTP binding in the absence of DNA. Next we solved the binary complex structures of Pol  in complex with 12 ACS Paragon Plus Environment

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MgdATP, MgdGTP or MgdCTP (Structures 5-7 in Table 1, with structural statistics shown in Tables S2). The diffraction pattern for the Pol MgdTTP binary complex was poor and the structure could not be obtained. The structures of the three binary complexes are very similar (Figure S5) as described below. As an example, Figure 1C compares the structures between Pol MndATP and Pol MgdATP binary complexes. As shown in the expanded structures (Figure 1D), while the nucleotide binding metal B and the catalytic metal A were both observed in the Pol :MndATP binary complex, the A-metal (catalytic metal) is missing in the Pol MgdATP binary complex. Analysis of the A-metal coordination site showed that Mg2+ failed to induce enough conformational changes to bring the relevant coordinating atoms to within the strict octahedron coordination distance (2.08 Å).45 For example, the closest distance between Asp418 side chain oxygen and the imaginary A-metal (borrowing the Mn2+ A-metal position from the Pol :MndATP binary complex) is 2.9 Å. Overall, only two out of the required six ligands exist, therefore, the overall coordination cannot fulfill the octahedron geometry for Mg2+. In addition, in the Pol MgdATP binary complex, the triphosphate tail of dATP and sidechains of His329 and Asp332 display dual conformations. In the conformation in which the triphosphate tail rotates away from the His329 and the B-metal, the interactions to His329 and to B-metal Mg2+ are lost. These multiple modes of MgdATP binding to Pol  suggest non-productive binding. Similarly, multiple modes of binding and the lack of A-metal were observed for the Pol MgdCTP binary complex and the Pol MgdGTP binary complex (Figure S5).



Figure 1. Structural comparison of Pol  binary complexes with MndNTP and MgdNTP in the absence of DNA. (A) Comparison of overall structures between apo Pol  (magenta, PDB code: 4LZD) and Pol :MndCTP binary complex (green). The active site residues involved in MndCTP binding and MndCTP are shown in stick with the zoom-in view shown in B. (B) Active site structural overlay of apo Pol  magenta) and Pol MndCTP binary complex (green) in which the carbon of dCTP are shown in yellow stick. Putative interactions are shown in green dash lines. Mn2+ metals A and B are shown in green sphere. (C) Active site structural overlay of Pol :MndATP binary complex (orange) and Pol :MgdATP (cyan) binary complex. For the Pol :MgdATP binary complex, two conformations were observed for the triphosphate (TP) moiety of dATP (indicated with red lines), H329 and D332. Heavy sticks were used for dATP in both complexes, Mn2+ in orange sphere and Mg2+ in cyan sphere. (D) Zoom-in figure of C with a slight rotation for clarity. The coordination water in the Pol :MndATP binary complex is shown in a blue sphere. The black dashed lines show the coordination and inter-atom distances to the Mn2+ A-metal in the Pol :MndATP binary complex, while the cyan dashed lines illustrate the failed octahedron coordination for the imaginary Mg2+ A-metal (borrowing the 14 ACS Paragon Plus Environment

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Mn2+ A-metal from the Pol :MndATP binary complex) in the Pol :MgdATP binary complex.

The Prebound MndNTP Adopts Similar Conformation to the Precatalytic Ternary Complex Pol :DNA:MndUMPNPP. For comparison, we then solved the structure of the precatalytic ternary complex of Pol :DNA:MndUMPNPP (Structure 8 in Table 1, with the structural statistics in Table S3). As shown in Figure 2A, the binding site conformation for the bound dCTP in the prebinding binary complex is very similar to that of the precatalytic ternary complex. For more detailed comparison, Figure 2B shows a schematic drawing to highlight some key interactions and distances (dashed lines a, b, and c), and Figure 2C shows the stereo view of the structural superimposition of apo Pol , Pol :MndCTP binary complex, Pol :DNA binary complex, and Pol :DNA:MndUMPNPP precatalytic ternary complex. It shows that His329 side chain rotates to form two critical hydrogen-bond interactions upon the incoming dUMPNPP binding to the Pol :DNA binary complex: one with the -phosphate of dUMPNPP (dashed line a) and the other with the primer terminal nucleotide (dashed line b).22 These interactions provided by His329 have been proposed to help position the primer terminus with the incoming dNTP when template nucleotides are missing.22 Trp434 also helps to stabilize the primer terminal sugar via -CH interactions in the precatalytic ternary complex as reported previously for other precatalytic ternary complexes.23,

59

Importantly, we found that these additive

conformational transitions for His329 and Trp434 induced together by DNA and the incoming dUMPNPP can be induced solely by MndCTP in the absence of DNA (Figure 2C). In addition, as shown in Figure 2C, the distance between the P of 15 ACS Paragon Plus Environment


dUMPNPP and the 3′O of the primer terminus in the Pol :DNA:MndUMPNPP precatalytic ternary complex is 3.5 Å (dashed line c), and similar distance (3.7 Å) is observed between the P of dCTP in the Pol :MndCTP binary complex to the 3′O of the primer terminus in the precatalytic ternary complex. These results together with the similar in-line geometry (Figure 2C) suggest that nucleophilic attack by the 3′O to the P of dCTP may take place when the Pol :MndCTP binary complex binds to the incoming DNA. Taken together, the results show that Pol  can be preactivated by MndNTP. For easy comparison, the distances of the dashed lines a-c for these complexes, and additional ones from later sections, are listed in Table S8.


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Figure 2. Structural comparison of Pol  precatalytic ternary complexes in the presence of 1 nt-gapped DNA. (A) Active site structural overlay of Pol MndCTP binary complex (green) with Pol DNA:MndUMPNPP precatalytic ternary complex (grey). (B) Schematic drawing of dNTP binding site to highlight the key interactions with indicated distances a, b and c to illustrate the productive MndNTP binary complex as shown in Figure C. (C) Structural overlay of apo Pol  (magenta), Pol :MndCTP binary complex (green), Pol :DNA binary (orange, PDB code: 4LZG) 17 ACS Paragon Plus Environment


and Pol :DNA:MndUMPNPP precatalytic ternary complex (grey). The distances (Å) between the P atom and the 3′O atom of the primer terminus are labeled. (D) Active site structural overlay of Pol DNA:MndUMPNPP precatalytic ternary complex (grey) and Pol DNA:MgdUMPNPP precatalytic ternary complex (cyan, PDB code: 4M04). (E) Active site structural overlay of Pol MgdCTP binary complex (magenta) with Pol :DNA:MgdUMPNPP precatalytic ternary complex (cyan, PDB code: 4M04). The dual triphosphate (TP) conformations of dCTP in the Pol MgdCTP binary complex are indicated with magenta lines.

Comparison of Mn2+ and Mg2+ Precatalytic Complexes. Interestingly, unlike the binary complexes, the precatalytic Pol :DNA:MndUMPNPP ternary complex is very similar to the corresponding Pol :DNA:MgdUMPNPP ternary complex reported previously.23 As shown in Figure 2D both ternary complexes contain the active conformations for catalysis as the distance between the 3′-OH oxygen of the primer terminal nucleotide and the phosphorus of -phosphate is 3.54 Å in both complexes (rows 1 and 6, Table S8), and the 3′-OH oxygen is in line with the phosphorus and the bridging oxygen atom of the pyrophosphate leaving group. Both metal A and metal B also superpose quite well between the two complexes. As a consequence, the conformation of MgdNTP in the prebinding binary complex is quite different from that in the corresponding precatalytic ternary complex Pol :DNA:MgdUMPNPP. As shown in Figure 2E, in the Pol MgdCTP binary complex, Mg2+ ion A is missing, and the triphosphate part of dCTP and His329 both adopt dual conformations, which are different from the active conformations of the precatalytic ternary complex.

Pol  can also be pre-activated by Mn·rNTP for DNA repair by NHEJ. 18 ACS Paragon Plus Environment

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Ribonucleotide incorporation and physiological concentration of Mn2+ ion have been shown to improve Pol ’s efficiency in gap-filling of NHEJ DNA substrates.54 By solving the structure of a precatalytic ternary complex of Pol  with a 1-nt gapped DNA and the non-hydrolyzable rUTP analog uridine-5'-[(α,β)-imido]triphosphate (UMPNPP), and compared it to the nonhydrolyzable dUTP (dUMPNPP) counterpart precatalytic ternary complex, Moon et al. showed that Pol  can incorporate ribonucleotide with normal active site geometry and without distortion in DNA or ribonucleotide.59 Here we exam whether MnrNTP or MgrNTP alone can induce similar active site conformation to that of the rNTP-containing precatalytic ternary complex. We first measured the affinity of MnrNTP and MgrNTP in the absence of DNA, and found that Pol  exhibits high affinity to MnrNTP with measured Kd values of 0.71-2.76 M, while the affinity toward MgrNTP is much weaker (Kd values of 98-236 M) (Figure S1 and Table 2). We then determined the structures of all four Pol :MnrNTP binary complexes where N is A, U, G or C (Structures 9-12 in Table 1, with the structural statistics in Table S4), as shown in Figure S6. As an example, Figure 3A shows that the conformation of the bound MnrATP is very similar to that of MndATP. As the structure of the Pol :DNA:MgUMPNPP precatalytic ternary complex is available (but not Pol :DNA:MnUMPNPP), we compare our Pol :MnrUTP binary complex to this structure. As shown in Figure 3B, the active site conformation induced by MnrUTP alone is very similar to that of the Pol :DNA:MgUMPNPP precatalytic ternary complex except the ribose and base moieties of rUTP and UMPNPP, as supported by the very similar distances a, b and c shown in Figure 3B and Table S8. 19 ACS Paragon Plus Environment


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Regarding the base conformations, similar to the Pol :dNTP binary complexes, due to the lack of a pi-pi interaction, the base conformations of rNTP appear to be diverse. The base of rUTP in the Pol :MnrUTP binary complex occupies the space for the primer terminus dA4 of DNA in the precatalytic ternary complex, suggesting a necessary movement of rUTP’s ring to free up space for dA4 upon DNA binding. Taken together, these results suggest that the ribonucleotide incorporation by Pol  can be preactivated by MnrNTP alone prior to DNA binding. In addition to the gap-filling function, another important function of Pol  in NHEJ is to add nucleotides to 3′ overhangs (ssDNA region) of broken DNA end to create microhomology for subsequent ligation.72 TdT also participates in this process.26 As both polymerases have the ability to incorporate rNTP, recently Pryor et al. tested the effect of rNTP incorporation on NHEJ, and found that addition of an rCTP to the 3′ end of a ssDNA overhang by Pol  or TdT increased the efficiency of the subsequent ligation step.26 The incorporated rCTP was subsequently excised rapidly by a ribonuclease.26 We are curious if binding of MnrNTP to Pol  can induce similar active site structure that is induced together by a ssDNA and a nucleotide. For this purpose, the structure of the TdT:ssDNA:MgddTTP precatalytic ternary complex73 is available for comparison (no precatalytic ternary complex of TdT containing a rNTP is available). Figure 3C shows the comparison of the active site structures

of

the

Pol

:MnrUTP

binary

complex

with

that

of

the

TdT:ssDNA:MgddTTP precatalytic ternary complex. Remarkably, the active site structures are very similar except the different conformations of the ribose and the base of rUTP and ddTTP, which suggests that the uridine ring of rUTP in the Pol 20 ACS Paragon Plus Environment

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:MnrUTP binary complex needs to rotate away to accommodate the terminal ddT6 nucleotide of the incoming DNA. Of particular interest is the interactions of His329 (His342 in TdT) with the -phosphate of the incoming rUTP and the plausible interaction with the terminal ddT6 nucleotide of the ssDNA. His329 in Pol  and His342 in TdT each plays a critical role in ssDNA extension as the Pol  H329A mutant is 40-fold less active than the WT enzyme,22 and the corresponding H342A mutant of TdT is 100-fold less active than the WT in the same function.22 Taken together, these results suggest that MnrNTP alone, in the absence of DNA, can also activate Pol  for its function in adding rNTP to a DNA overhang in NHEJ.26



Figure 3. Structural properties of Pol Mn·nucleotide complexes for different functions. (A) Active site structural overlay of Pol :MnrATP (magenta) and 22 ACS Paragon Plus Environment

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Pol :MndATP (marine). (B) Structural overlay of Pol :MnrUTP binary complex (green) and Pol :DNA:MgUMPNPP precatalytic ternary complex (grey, PDB code: 5TWP). The interactions between atoms are shown in dashed lines with the colors matching those of the structures, DNA primer in cartoon and the primer terminal nucleotide in line. Distances a, b and c are shown. Note that Trp434 adopts two conformations in the precatalytic ternary complex, and only the major form is displayed here. (C) Structural overlay of the Pol :MnrUTP binary complex (green) and the TdT:ssDNA:MgddTTP precatalytic ternary complex (protein side chains and ddTPP in magenta, DNA in grey, PDB code: 4I27). Both of the A and B metal ions are Mn2+ for the Pol :MnrUTP binary complex, and the B-metal is Mg2+ for the TdT ternary complex. For simplicity, the residue numbers of TdT are not labeled in the figure. The corresponding residues are listed here in the form of TdT(Pol ): R336(R323), K338(K325), H342(H329), D343(D330), D345(D332), D434(D418) and W450(W434). (D) Structural overlay of the Pol :MndTTP binary complex (cyan) and the TdT:ssDNA:MgddTTP precatalytic ternary complex (the same complex as in figure C).

Pol  can be pre-activated by Mn·dNTP for its terminal transferase TdT-like function. Pol  possesses terminal transferase (template-independent) TdT-like activity in adding deoxynucleotides to a ssDNA.21-22, 27, 74 For this function of Pol , Mn2+ ion instead of Mg2+ is required21-22, 27, 74, however, there has not been any structural basis explanation for this unique Mn2+ specificity. We found that this Mn2+ ion preference over Mg2+ ion is consistent with our ITCs results that show high affinity of Pol  to MndNTP but very weak affinity to MgdNTP in the absence of DNA (Table 2). In terms of metal ion, TdT is not specific to Mn2+ and it utilizes divalent metal ions in the preferred order of Mg2+, Zn2+, Co2+ and Mn2+.75-76 As the TdT-like function is template-independent, and the affinity of MndNTP (Kd values in the range of 0.22-1.95 M, Table 2) is higher than that of a ssDNA to Pol  (Kd = 23 ACS Paragon Plus Environment


11.05±1.31 M, Table 2), we are curious if Pol  can be preactivated by MndNTP alone for its TdT-like terminal transferase activity. As no structures of Pol  complexes containing a ssDNA are available, we compare the structure of our Pol MndTTP binary complex to that of the TdT:ssDNA:MgddTTP precatalytic ternary complex. As shown in Figure 3D, similar to the case of the Pol MnrUTP binary complex shown in Figure 3C, the active site structures in these two complexes are very similar except the ribose and the base of dTTP and ddTTP (see below). The high similarity in the active sites therefore suggests that Pol  can be preactivated by MndNTP for its terminal transferase activity prior to ssDNA binding. The structural comparison in Figure 3D suggests that the thymine ring of dTTP in the Pol :MndTTP binary complex needs to rotate away to make room for the terminal ddT6 nucleotide of the incoming ssDNA.

Comparison between the Structures of Pol μ:Mn8-oxo-dGTP and Pol μ:MndGTP Binary Complexes. Due to its dual coding potential, 8-oxo-dGTP is mutagenic once incorporated into a genome. Despite its previous studies in 8-oxo-G bypass,77 Pol  has never been shown to incorporate 8-oxo-dGTP into DNA until a recent report of functional studies during the preparation of this paper.65 This situation provided us with a good opportunity to perform a full spectrum of structural study of Pol  with this mutagenic nucleotide analogue, from its prebinding ability to its precatalytic and postcatalytic states. As shown in Table 2, Pol  can prebind Mn8-oxo-dGTP (Kd = 6.7±1.2 M) but only weakly to Mg8-oxo-dGTP (Kd = 191±8 M). We solved the structure of Pol  in 24 ACS Paragon Plus Environment

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complex with Mn8-oxo-dGTP (Structure 13 in Table 1, with structural statistics in Table S5). The structure of Pol  in complex with Mg8-oxo-dGTP could not be solved due to poor diffraction. Figure 4A shows that both Mn8-oxo-dGTP and MndGTP bind to Pol  in an anti conformation, and form a pi-pi interaction with Trp434 side chain. In both cases, the -phosphate forms a H-bond with His329 side chain. In addition, the triphosphate moiety and the two Mn2+ ions are almost superimposable between the two complexes. One difference is that the ribose adopts a C2-endo conformation in 8-oxo-dGTP while it adopts a C3-endo in dGTP, which is an expected and commonly observed conformational change in polymerase catalysis upon formation of a catalytic ternary complex.62,

78-79

These very similar structures

between the Pol :Mn8-oxo-dGTP and the Pol :MndGTP binary complexes strongly suggest that Pol  can also be pre-activated by Mn8-oxo-dGTP prior to DNA binding.

Structures of Precatalytic Complexes for 8-oxo-dGTP Incorporation by Pol . To further compare the Pol :Mn8-oxo-dGTP binary complex with a precatalytic ternary complex containing 8-oxo-dGTP, we used Ca2+ ion which does not support catalysis,30,

78

and solved the structures of the precatalytic ternary complexes with

both dA template and dC template (Structures 14 and 15, respectively, in Table 1, with structural statistics in Table S6). The overlaid structures of the Pol :Mn8-oxo-dGTP binary complex and the Pol :dA:Ca8-oxo-dGTP precatalytic ternary complex (Figure 4B) show high similarity in the active site protein side chain conformations, the divalent metal binding, and the triphosphate part of 8-oxo-dGTP. An exception in the structural similarity is the conformation of 8-oxo-dGTP. In the 25 ACS Paragon Plus Environment


Pol :dA:Ca8-oxo-dGTP precatalytic ternary complex, 8-oxo-dGTP adopts a syn conformation to pair up with the template dA, and forms an anti:syn dA:8-oxo-dGTP base pair (Figure 4B), while in the Pol :Mn8-oxo-dGTP binary complex, the prebound 8-oxo-dGTP adopts an anti conformation. This suggests that the anti 8-oxo-dGTP needs to rotate to a syn conformation upon binding of the template dA-containing DNA. This rotation can also free up space to accommodate the primer terminus dA4 of the incoming DNA. Similarly, the overlaid structures of the Pol :Mn8-oxo-dGTP binary complex and the Pol :dC:Ca8-oxo-dGTP (anti:anti) precatalytic ternary complex show high structural similarity except the ribose and base moieties of 8-oxo-dGTP (Figure 4C). In the Pol :Mn8-oxo-dGTP binary complex, the base of 8-oxo-dGTP occupies the space for the primer terminus dA4 of the incoming DNA. Therefore, the base of 8-oxo-dGTP needs to rotate away to free up the space, and also to form a base pair with the template dC of the incoming DNA to form an anti:anti dC:8-oxo-dGTP base pair. Similar to the analysis in Pol :MndNTP and Pol :MnrNTP binary complexes, the measured P-3′O distances (rows 11-13, Table S8) support that the nucleophilic attack may occur upon DNA binding to the Pol :Mn8-oxo-dGPT binary complex. Taken together, the results indicate that the active site conformations of the Pol :dA:Ca8-oxo-dGTP (anti:syn) and Pol :dC:Ca8-oxo-dGTP (anti:anti) precatalytic ternary complexes are both similar to that of the Pol :Mn8-oxo-dGTP binary complex except the guanine moiety of 8-oxo-dGTP, which is expected to undergo a conformational adjustment upon binding of DNA to the Pol :Mn8-oxo-dGTP binary complex. These results support that Mn8-oxo-dGTP alone can activate Pol  for formation of the precatalytic ternary 26 ACS Paragon Plus Environment

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complex with a mismatched (dA template) or matched (dC template) DNA.

Figure 4. Structures of Mn8-oxo-dGTP binding and incorporation at different states. (A) Overlay of Pol :Mn8-oxo-dGTP binary complex (protein in magenta, 8-oxo-dGTP in heavy magenta stick, Mn2+ metal ions A and B in magenta sphere) and Pol :MndGTP binary complex (protein in green, 8-oxo-dGTP in heavy green stick, Mn2+ metal ions A and B in green spheres. (B) Stereo view comparison of the Pol :Mn8-oxo-dGTP binary complex (green, Mn2+ metal ions A and B in green sphere) and the Pol : dA:Ca8-oxo-dGTP precatalytic ternary (protein in magenta, Ca2+ metal ions A and B in magenta sphere, DNA in grey lines, 8-oxo-dGTP in heavy grey stick). The distances (Å) between the P atom and the 3′O atom of the primer terminus are labeled. (C) Stereo view comparison of the Pol :Mn8-oxo-dGTP binary complex (green) and the Pol :dC:Ca8-oxo-dGTP precatalytic ternary (protein in grey, Ca2+ metal ions A and B in grey spheres, DNA in yellow lines, 8-oxo-dGTP in 27 ACS Paragon Plus Environment


heavy yellow stick).

Structures of Product Complexes from Mn2+ and Mg2+ Mediated Incorporation of 8-oxo-dGTP. We next solved the structures of the product complexes by soaking the precatalytic ternary complex crystals with 10 mM MnCl2 or MgCl2 for 16 hrs (Structures 16-19 in Table 1, with structural statistics in Table S7). We observed the incorporated product complex containing a dA:8-oxo-dGMP (anti:syn) Hoogsteen base pair from both MnCl2 and MgCl2 soaking (Structures 16 and 17, respectively, superimposed in Figure 5A), and the incorporated product complex dC:8-oxo-dGMP (anti:anti) W-C base pair also from both MnCl2 and MgCl2 soaking (Structures 18 and 19, respectively, superimposed in Figure 5B). In both 5A and 5B, the two structures including metal ions A and B superimpose quite well, except the following minor differences: (i) In Structure 17, approximately 30% His329 retains in the ternary state, suggesting that this part of protein has not fully turned to the product state. (ii) Structure 18 contains two additional metals named metal C and metal D. Metal D serves to neutralize negative charge by interacting with one of the phosphodiester oxygen atoms (Figure 5C). Metal C interacts with one of the -oxygens of the leaving pyrophosphate, the 8-oxo oxygen, the 5'-oxygen and one of the non-bridging phosphodiester oxygen atoms of the incorporated 8-oxo-dGMP (Figure 5C). This stabilization of anti 8-oxo-dGMP by a product metal has also been observed for Pol .62 (iii) The state of the pyrophosphate (PPi) product differs. In Structure 16, it has left and its vacancy is occupied by oxalate. In structures 17 and 19, it appears to have been hydrolyzed to a single phosphate group, a property that has 28 ACS Paragon Plus Environment

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been reported recently,80 while in Structure 18 the PPi is retained. (iv) In Structure 19, Asp330 appears to sample two conformations. Taken together the results of Pol  described here and the results of Pol 62 and Pol 63 reported recently, the fine structures of the metal ions and the PPi are highly variable with specific conditions in the product complexes of 8-oxo-dGTP incorporation.



Figure 5. Structures of 8-oxo-dGMP product complexes. (A) Comparison between the Pol :dA:Mn8-oxo-dGMP product complex (Structure 16) (cyan) and the Pol :dA:Mg8-oxo-dGMP product complex (Structure 17) (magenta). Mn2+ ions A and B are shown in cyan sphere, and Mg2+ ions A and B in magenta spheres. For simplicity, the upstream base pairs are shown in lines. (B) Comparison between the Pol :dC:Mn8-oxo-dGMP product complex (Structure 18) (grey, and the PPi group in wheat) and the Pol :dC:Mg8-oxo-dGMP product complex (Structure 19) (green). In the Pol :dC:Mn8-oxo-dGMP product complex, additional metal C and D are also observed. (C) Expanded active site region of Structure 18 (in a different view for 30 ACS Paragon Plus Environment

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clarity) showing the four metal ion coordination. Water molecules are shown in blue spheres, and metal coordination in light blue lines.

Discussion

Major Advances of this Study. By solving 19 crystal structures of Pol  and its complexes, we show that the noncanonical mechanism involving prebinding of MndNTP is relevant to the various functions of Pol  - dNTP incorporation into a 1-nt gapped DNA, rNTP incorporation for NHEJ, dNTP addition to a ssDNA in TdT-like activity, and 8-oxo-dGTP incorporation into mismatched (dA) or matched (dC) template. In the presence of Mg2+ ion, Pol  follows the canonical pathway of binding DNA first, then MgdNTP (Figure 6). Previously this noncanonical mechanism was suggested to be operating in parallel to the canonical mechanism for ASFV Pol X16 and human Pol  The noncanonical mechanism enables ASFV Pol X to prebind MgdGTP before DNA and catalyzes a dG:dGTP mismatch16, resulting in its extremely low fidelity value of 2. For Pol  with moderate fidelity, the noncanonical mechanism further reduced the fidelity of the L431A mutant which has enhanced MgdNTP prebinding propety.17 For Pol , we previously reported that binding of MgdNTP in the absence of DNA was too weak to be measured by ITC, indicating that the binding is too weak to be functionally relevant. Therefore, Pol  strictly follows the canonical pathway of DNA binding first, then MdNTP, enables its high fidelity property (near 100,000)11. These properties are illustrated in Figure 6. Taken together with previous reports, we propose that different DNA polymerases in the X-family can use one or a 31 ACS Paragon Plus Environment


combination of the canonical and the noncanonical mechanisms to achieve their desired biological functions and fidelity.

Figure 6. Reaction pathways of X-family Pols. In the presence of Mn2+, Pol  is capable of binding dNTP, rNTP or 8-oxo-dGTP productively, before binding DNA (noncanonical). In the presence of Mg2+ ion, Pol  can follow the canonical pathway. Based on previous studies,16-17 Pol  and ASFV Pol X are also capable of catalysis via the noncanonical reaction pathway (in addition to the canonical pathway), while Pol  follows strictly the canonical pathway.

Implication on the Past Studies of Pol . In the seminal work on Pol  almost two decades ago, Dominguez and coworkers showed that for a primer-template DNA extension reaction, in the presence of Mg2+ ion, Pol  preferentially incorporated the complementary deoxynucleotides. However, in the presence of Mn2+ ion, Pol  behaves like a mutator polymerase by incorporating deoxynucleotides in a random manner with very poor base selectivity.21 This long-standing puzzle of mutator property caused by Mn2+ ion may now be explained by the unique MndNTP-prebinding and preactivation property discovered in this study. In the presence of Mn2+ ion, each of the four dNTPs can prebind Pol  in a catalysis-ready conformation. Upon DNA binding, the prebound dNTP then forms a base pair with the template base. This binding order of MndNTP first then DNA overcomes the Watson-Crick base pairing rule, and results in the random incorporation pattern. On 32 ACS Paragon Plus Environment

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the other hand, in the presence of Mg2+ ion, MgdNTP-prebinding is disfavored (Table 2), and Pol  follows the canonical pathway. Pol  has been shown to incorporate more rNTP with Mn2+ than with Mg2+ in a highly distributive manner.57 This may also be explained by the MnrNTP preactivation property of Pol  discovered in this work. Moon et al. proposed that, when the dNTP concentrations are at its lowest level during the G1 phase of cell cycle and in nonreplicating cells, the rapid incorporation of rNTP by Pol  which could be rapidly removed via ribonucleotide excision repair, may help to combat the tremendous DSBs threat to the genome integrity.59 Based on the MnrNTP preactivation, it is likely that Pol  is already preloaded with each of the four MnrNTP and can add them to the DNA when it binds to the Ku-DNA complex. For the template-independent TdT-like reaction of Pol  in adding nucleotides to a ssDNA, MnCl2 has been used.22, 74 The requirement of Mn2+ ion instead of Mg2+ in the template-independent reaction can now be understood by our MndNTP preactivation mechanism. In the presence of Mn2+, Pol  can be preloaded with MndNTP or MnrNTP, therefore it can add them to a ssDNA. On the other hand, binding of MgdNTP requires the instruction from the template base, which is lacking in a ssDNA substrate. Finally, for Pol  to proceed via the noncanonical pathway, it requires dissociation of DNA upon completion of the catalytic cycle. The distributive nature of Pol  has been well-documented.21, 56-57, 81-82 Consistently, we show that Pol  binds MndNTP or MnrNTP more tightly than a ssDNA, and more tightly or similarly than a 1-nt gapped DNA. 33 ACS Paragon Plus Environment


Incorporation of 8-oxo-dGTP by Pol . In addition, we show that, like other members of the X-family Pol  and Pol , Pol  can also incorporate 8-oxo-dGTP into DNA in both dA:8-oxo-dGTP (anti:syn) Hoogsteen and dC:8-oxo-dGTP (anti:anti) Watson-Crick pairs. Again, Pol  is able to prebind Mn8-oxo-dGTP but not Mg8-oxo-dGTP in an active conformation. During the preparation of this manuscript, a report by Çağlayan and Wilson showed that, in a coupled DNA polymerase and DNA ligase assay, Pol  in the presence of MgCl2 not only can insert 8-oxo-dGTP opposite a template dA or dC, it can also insert 8-oxo-dGTP opposite a template dT with an even higher efficiency.65

Two Remaining Questions for the Feasibility of the Noncanonical Pathway. The first question is whether MndNTP can still be competitive with MgdNTP considering the physiological concentrations of the two divalent ions. The reported physiological Mn2+ concentration is in the range of 20–53 M,54 and in the range of 60–150 M in the brain,83 but it can be higher than these values with overexposure of Mn.47-49 It has also been found that with Mn2+ exposure to brain cells, the majority of intracellular Mn2+ was found to be present in the nucleus.84-85 Concentration of 0.8-1.2 mM free Mg2+ have been measured in cardiac and liver mitochondria,86-87 and similar free Mg2+ in nucleus is assumed based on the porous structure of the nuclear envelope.88 Overall, if we compare the concentration range between Mg2+ (0.8-1.2 mM) and Mn2+ (20–53 M), the concentration of Mg2+ in the nucleus is ca. 23-40 fold higher than that of Mn2+, while the Kd value of MgdNTP is roughly two orders higher 34 ACS Paragon Plus Environment

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than that of MndNTP (Table 2). Thus, MndNTP is in principle more favored than or competitive with MgdNTP, though the relative competitiveness will depend on specific cellular compartments and other cellular factors. Furthermore, the in vivo concentrations of dNTP in a dividing cell have been reported to be in the range of 5– 50 M,89 while the concentrations of rNTP are 40-350 fold and 160-2000 fold higher in cycling cells and confluent cells, respectively.89-90 Therefore, the cellular concentrations of dNTP and rNTP are both higher than the Kd values of MndNTP to Pol  (ranging from 0.22 ± 0.02 M to 1.95 ± 0.42 M, Table 2). On the other hand, the concentration of damaged DNA is presumably much lower than that of MndNTP or MnrNTP, therefore the MndNTP/MnrNTP prebinding should be possible for Pol  in vivo. The other question is that, even though DNA (particularly damaged DNA) is highly disfavored thermodynamically to compete with MndNTP for binding the apo Pol  as described above, can it really bind to the Pol :MndNTP binary complex without requiring the bound MndNTP to dissociate? This is difficult to demonstrate since in solution the enzyme, metal ion, dNTP, and DNA are in microscopic equilibrium. In crystals, DNA could not be soaked into Pol :MndNTP, but nor to apo Pol . Our confidence in the feasibility of the proposed noncanonical pathway for Pol /Mn2+ is based on several factors: it is favored thermodynamically, feasible in vivo, relevant functionally, and demonstrated structurally. Furthermore, a thorough demonstration of this feasibility has also been demonstrated previously for ASFV Pol X,16, 91 though only for MgdGTP specifically.



Conclusion. This work describes two significant findings based on 19 crystal structures of Pol : (1) The noncanonical mechanism becomes the main pathway for various functions of Pol . This should make the “noncanonical mechanism” no longer noncanonical. (2) The noncanonical mechanism is operating only with the noncanonical (but functionally preferred) divalent metal Mn2+ instead of Mg2+. This solved the long standing puzzle in the Mn2+ preference of Pol . Taken together with the properties of three other pols in the X-family, we can conclude that high fidelity and replicative DNA polymerases bind DNA first followed by MgdNTP, while low fidelity and repairing DNA polymerases may bind preferred metal-nucleotide first followed by DNA binding, as illustrated in Figure 6.

Acknowledgments: This work is supported with funds from the Ministry of Science and Technology (Grant No. MOST103-2113-M-001-016-MY3), and the Taiwan Protein Project, Academia Sinica (Grant No. AS-KPQ-105-TPP) to M.-D.T. We thank Dr. A. Moon for providing the Pol μ Δ2 construct. We thank the technical services provided by the “Synchrotron Radiation Protein Crystallography Facility of the National Core Facility Program for Biotechnology, Ministry of Science and Technology” and the “National Synchrotron Radiation Research Center”, a national user facility supported by the Ministry of Science and Technology of Taiwan, ROC. The synchrotron radiation experiments in Japan were performed at the BL32XU of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2017A2576, 2017B2576, and 2018A2514). We thank Dr. Shu-Chuan (Chris) Jao and 36 ACS Paragon Plus Environment

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Ms Szu-Huan Wang of the Biophysics Core Facility, Department of Academic Affairs and Instrument Service at Academia Sinica for providing support with data acquisition of ITC experiments. We thank Dr. Meng-Chiao Ho for helpful discussions.

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DNA Polymerase X from African Swine Fever Virus. Biochemistry 2008, 47 (30), 7875-7887.

Table of Contents graphic:


Human DNA Polymerase Î¼ Can Uses a Noncanonical Mechanism for

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