Biomedical subjects

Shigenori Iwai

Publications and source records attributed to Shigenori Iwai.

At least 19 recordsLinked to original sources

PubMed2026

Overlapping RAD18- and DNA-binding interfaces in DNA polymerase η contribute to UV-induced DNA damage tolerance.

DNA polymerase η (Polη) bypasses UV-induced pyrimidine dimers and thereby confers tolerance to UV irradiation. Although the C-terminus of Polη has been reported to interact with ubiquitinated PCNA and RAD18, how Polη engages RAD18 is not fully understood. Here, we show that Polη and RAD18 interact through two distinct modes in human cells: a ubiquitinated-PCNA-dependent mode that requires the Polη C-terminus, and an unexpected PCNA-independent mode mediated by its N-terminal region. We focused our subsequent analyses on this newly identified PCNA-independent mode. Using purified recombinant proteins, we demonstrate direct binding of the N-terminal region of human Polη (PolηΔC) to RAD18 in vitro. Although PolηΔC and RAD18 each bound primer-template DNA, we were unable to detect a ternary PolηΔC-RAD18-DNA complex, and DNA competitively inhibited RAD18 binding to both PolηΔC and full-length Polη. Mutational analyses revealed that the DNA-binding and RAD18-binding domains within Polη overlap. A separation-of-function mutant, PolηΔC(K317A), which retains near-normal DNA-binding and polymerase activities but exhibits reduced RAD18 binding in vitro, displayed a diminished ability to rescue the UV sensitivity of Polη-deficient cells. Notably, the detrimental impact of the K317A persisted in a PCNA-binding-defective background but was attenuated in RAD18-knockout cells. These findings demonstrate that RAD18 binding to the N-terminal domain of Polη contributes to efficient bypass of pyrimidine dimers independently of the Polη-PCNA interaction and provide mechanistic insights into how Polη-RAD18 complexes assemble and dissociate during translesion DNA synthesis.

Journal Article↗

PubMed2007

The intricate structural chemistry of base excision repair machinery: implications for DNA damage recognition, removal, and repair.

Three-dimensional structures of DNA N-glycosylases and N-glycosylase/apyrimidine/apurine (AP)-lyase enzymes and other critical components of base excision repair (BER) machinery including structure-specific nuclease, repair polymerase, DNA ligase, and PCNA tethering complexes reveal the overall unity of the simple cut and patch process of DNA repair for damaged bases. In general, the damage-specific excision is initiated by structurally-variable DNA glycosylases targeted to distinct base lesions. This committed excision step is followed by a subsequent damage-general processing of the resulting abasic sites and 3' termini, the insertion of the correct base by a repair DNA polymerase, and finally sealing the nicked backbone by DNA ligase. However, recent structures of protein-DNA and protein-protein complexes and other BER machinery are providing a more in-depth look into the intricate functional diversity and complexity of maintaining genomic integrity despite very high levels of constant DNA base damage from endogenous as well as environmental agents. Here we focus on key discoveries concerning BER structural biology that speak to better understanding the damage recognition, reaction mechanisms, conformational controls, coordinated handoffs, and biological activities including links to cancer. As the three-dimensional crystal and NMR structures for the protein and DNA complexes of all major components of the BER system have now been determined, we provide here a relatively complete description of the key complexes starting from DNA base damage detection and excision to the final ligation process. As our understanding of BER structural biology and the molecular basis for cancer improve, we predict that there will be multiple links joining BER enzyme mutations and cancer predispositions, such as now seen for MYH. Currently, structural results are realizing the promise that high-resolution structures provide detailed insights into how these BER proteins function to specifically recognize, remove, and repair DNA base damage without the release of toxic and mutagenic intermediates.

Animals↗

PubMed2006

Chemical synthesis of oligodeoxyribonucleotides containing the Dewar valence isomer of the (6-4) photoproduct and their use in (6-4) photolyase studies.

The pyrimidine(6-4)pyrimidone photoproduct, a major UV lesion formed between adjacent pyrimidine bases, is transformed to its Dewar valence isomer upon exposure to UVA/UVB light. We have synthesized a phosphoramidite building block of the Dewar photoproduct formed at the thymidylyl(3'-5')thymidine site and incorporated it into oligodeoxyribonucleotides. The diastereoisomers of the partially protected dinucleoside monophosphate bearing the (6-4) photoproduct, which were caused by the chirality of the phosphorus atom, were separated by reversed-phase chromatography, and the (6-4) photoproduct was converted to the Dewar photoproduct by irradiation of each isomer with Pyrex-filtered light from a high-pressure mercury lamp. The Dewar photoproduct was stable under both acidic and alkaline conditions at room temperature. After characterization of the isomerized base moiety by NMR spectroscopy, a phosphoramidite building block was synthesized in three steps. Although the ordinary method could be used for the oligonucleotide synthesis, benzimidazolium triflate as an alternative activator yielded better results. The oligonucleotides were used for the analysis of the reaction and the binding of Xenopus (6-4) photolyase. Although the affinity of this enzyme for the Dewar photoproduct-containing duplex was reportedly similar to that for the (6-4) photoproduct-containing substrate, the results suggested a difference in the binding mode.

Animals↗

PubMed2006

Human DNA polymerase N (POLN) is a low fidelity enzyme capable of error-free bypass of 5S-thymine glycol.

Human DNA polymerase N (POLN or pol nu) is the most recently discovered nuclear DNA polymerase in the human genome. It is an A-family DNA polymerase related to Escherichia coli pol I, human POLQ, and Drosophila Mus308. We report the first purification of the recombinant enzyme and examination of its biochemical properties, as a step toward understanding the functions of POLN. Unusual for an A-family DNA polymerase, POLN is a low fidelity enzyme incorporating T opposite template G with a frequency of 0.45 and G opposite template T with a frequency of 0.021. The frequency of misincorporation of T opposite template G is higher than any other known DNA polymerase. POLN has a processivity of DNA synthesis (1-100 nucleotides) similar to the exonuclease-deficient Klenow fragment of E. coli pol I, is inhibited by dideoxynucleotides, and resistant to aphidicolin. The strand displacement activity of POLN was higher than exonuclease-deficient Klenow fragment. Furthermore, POLN can perform translesion synthesis past thymine glycol, a common endogenous and radiation-induced product of reactive oxygen species damage to DNA. Thymine glycol blocks DNA synthesis by most DNA polymerases, but POLN was particularly adept at efficient and accurate translesion synthesis past a 5S-thymine glycol.

Amino Acid Sequence↗

PubMed2006

The GT to GC single nucleotide polymorphism at the beginning of an alternative exon 2C of human MTH1 gene confers an amino terminal extension that functions as a mitochondrial targeting signal.

Human MTH1 protein hydrolyzes oxidized purine nucleotides 8-oxo-2'-deoxyguanosine triphosphate (8-oxo-dGTP), 2-OH-dATP or their ribo-forms to their monophosphates, thus minimizing replicational and transcriptional errors both in the nuclei and mitochondria. MTH1 suppresses mitochondrial dysfunction and cell death caused by H(2)O(2). Furthermore, MTH1 suppresses the transient increase in 8-oxoguanine in mitochondrial DNA in the dopaminergic nerve terminals in mouse striatum after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration, and it protects the nerve terminals. We previously reported that a novel MTH1 allele with a single nucleotide polymorphism (SNP) in its exon 2c segment encodes the fourth MTH1 isoform, namely, MTH1a (p26), in addition to the three known isoforms, MTH1b (p22), c (p21), and d (p18). Another SNP located in exon 4 of the MTH1 gene, which is closely linked to the SNP in exon 2c, substitutes the Val83 residue in MTH1d with Met83. We herein show that all MTH1 isoforms efficiently hydrolyzed 2-OH-dATP and 8-oxo-dGTP. The amino terminal region of MTH1a functioned as a mitochondrial targeting signal when it was expressed in the HeLa cells as a fusion protein with enhanced green fluorescent protein. The cellular fractionation revealed that MTH1a(Met83) was localized in the mitochondria to the same extent as was MTH1d(Val83). However, the mitochondrial translocation of MTH1d(Met83) was less efficient than that of MTH1d(Val83).

Adenosine Triphosphate↗

PubMed2006

Conserved XPB core structure and motifs for DNA unwinding: implications for pathway selection of transcription or excision repair.

The human xeroderma pigmentosum group B (XPB) helicase is essential for transcription, nucleotide excision repair, and TFIIH functional assembly. Here, we determined crystal structures of an Archaeoglobus fulgidus XPB homolog (AfXPB) that characterize two RecA-like XPB helicase domains and discover a DNA damage recognition domain (DRD), a unique RED motif, a flexible thumb motif (ThM), and implied conformational changes within a conserved functional core. RED motif mutations dramatically reduce helicase activity, and the DRD and ThM, which flank the RED motif, appear structurally as well as functionally analogous to the MutS mismatch recognition and DNA polymerase thumb domains. Substrate specificity is altered by DNA damage, such that AfXPB unwinds dsDNA with 3' extensions, but not blunt-ended dsDNA, unless it contains a lesion, as shown for CPD or (6-4) photoproducts. Together, these results provide an unexpected mechanism of DNA unwinding with implications for XPB damage verification in nucleotide excision repair.

Amino Acid Motifs↗

PubMed2006

Synthesis and characterization of oligonucleotides containing 2'-fluorinated thymidine glycol as inhibitors of the endonuclease III reaction.

Endonuclease III (Endo III) is a base excision repair enzyme that recognizes oxidized pyrimidine bases including thymine glycol. This enzyme is a glycosylase/lyase and forms a Schiff base-type intermediate with the substrate after the damaged base is removed. To investigate the mechanism of its substrate recognition by X-ray crystallography, we have synthesized oligonucleotides containing 2'-fluorothymidine glycol, expecting that the electron-withdrawing fluorine atom at the 2' position would stabilize the covalent intermediate, as observed for T4 endonuclease V (Endo V) in our previous study. Oxidation of 5'- and 3'-protected 2'-fluorothymidine with OsO4 produced two isomers of thymine glycol. Their configurations were determined by NMR spectroscopy after protection of the hydroxyl functions. The ratio of (5R,6S) and (5S,6R) isomers was 3:1, whereas this ratio was 6:1 in the case of the unmodified sugar. Both of the thymidine glycol isomers were converted to the corresponding phosphoramidite building blocks and were incorporated into oligonucleotides. When the duplexes containing 2'-fluorinated 5R- or 5S-thymidine glycol were treated with Escherichia coli endo III, no stabilized covalent intermediate was observed regardless of the stereochemistry at C5. The 5S isomer was found to form an enzyme-DNA complex, but the incision was inhibited probably by the fluorine-induced stabilization of the glycosidic bond.

Deoxyribonuclease (Pyrimidine Dimer)↗

PubMed2006

Preferential formation of (5S,6R)-thymine glycol for oligodeoxyribonucleotide synthesis and analysis of drug binding to thymine glycol-containing DNA.

We previously reported the chemical synthesis of oligonucleotides containing thymine glycol, a major form of oxidative DNA damage. In the preparation of the phosphoramidite building block, the predominant product of the osmium tetroxide oxidation of protected thymidine was (5R,6S)-thymidine glycol. To obtain the building block of the other isomer, (5S,6R)-thymidine glycol, in an amount sufficient for oligonucleotide synthesis, the Sharpless asymmetric dihydroxylation (AD) reaction was examined. Although the reaction was very slow, (5S,6R)-thymidine glycol was obtained in preference to the (5R,6S) isomer. The ratio of (5S,6R)- and (5R,6S)-thymidine glycols was 2:1, and a trans isomer was also formed. When an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate, was used as a co-solvent, the reaction became faster, and the yield was improved without changing the preference. The phosphoramidite building block of (5S,6R)-thymidine glycol was prepared, and oligonucleotides containing 5S-thymine glycol were synthesized. One of the oligonucleotides was used to analyze the binding of distamycin A to thymine glycol-containing DNA by Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements. Distamycin A bound to a duplex containing either isomer of thymine glycol within the AATT target site, and its binding was observed even when the thymine glycol was placed opposite cytosine.

Anti-Bacterial Agents↗

PubMed2006

Chemical synthesis of oligonucleotides containing damaged bases for biological studies.

Since nucleic acids are organic molecules, even DNA, which carries genetic information, is subjected to various chemical reactions in cells. Alterations of the chemical structure of DNA, which are referred to as DNA damage or DNA lesions, induce mutations in the DNA sequences, which lead to carcinogenesis and cell death, unless they are restored by the repair systems in each organism. Formerly, DNA from bacteria and bacteriophages and DNA fragments treated with UV or gamma radiation, alkylating or crosslinking agents, and other carcinogens were used as damaged DNA for biochemical studies. With these materials, however, it is difficult to understand the detailed mechanisms of mutagenesis and DNA repair. Recent progress in the chemical synthesis of oligonucleotides has enabled us to incorporate a specific lesion at a defined position within any sequence context. This method is especially important for studies on mutagenesis and translesion synthesis, which require highly pure templates, and for the structural biology of repair enzymes, which necessitates large amounts of substrate DNA as well as modified substrate analogs. In this review, the various phosphoramidite building blocks for the synthesis of lesion-containing oligodeoxyribonucleotides are described, and some examples of their applications to molecular and structural biology are presented.

Cross-Linking Reagents↗

PubMed2006

Synthesis of oligonucleotides containing the Dewar valence isomer of the (6-4) photoproduct and their application to (6-4) photolyase studies.

The (6-4) photoproduct, which is a major UV light-induced lesion formed between adjacent pyrimidine bases, is isomerized to its Dewar valence isomer by exposure to longer wavelengths. We have synthesized a phosphoramidite building block of the Dewar photoproduct formed at the thymidylyl(3'-5')thymidine site, and incorporated it into oligonucleotides on a DNA synthesizer, aiming to use them for biological studies. An alternative activator, benzimidazolium triflate, gave better results, while by-products were detected at longer retention time in the ordinary synthesis. We characterized the synthetic oligonucleotides by UV conversion, nuclease digestion and mass spectrometry. Their use in the study of the (6-4) photolyase, a DNA repair enzyme, revealed its different recognition modes between the (6-4) photoproduct and the Dewar isomer.

Chromatography, High Pressure Liquid↗

PubMed2006

Characterization of the binding of distamycin A to damaged DNA: a comparison with the DNA recognition of the human DDB protein.

Distamycin A binds to DNA containing the (6-4) photoproduct, a major UV lesion that is recognized by the damaged DNA-binding (DDB) protein in human cells. We analyzed the binding properties of distamycin A and compared the results with those of the DDB protein. Structural change of the DNA duplex was not observed for distamycin A in two types of experiments, whereas the protein induced a large bending of the helix. Although the substrate specificity was different between the drug and the protein, thymine glycol was recognized by both of them, and inhibition of the DDB protein binding to the (6-4) photoproduct-containing DNA by distamycin A was tested.

Binding Sites↗

PubMed2006

Structural analysis of DNA containing (6-4) photoproduct in complex with distamycin A by NMR.

We reported that distamycin A can bind to DNA containing the (6-4) photoproduct, one of the major UV lesions in DNA, by means of CD spectroscopy. Here, we have analyzed the structure of DNA containing the (6-4) photoproduct in complex with distamycin A by NMR. Broadening of NMR resonances and large chemical shift perturbation have been observed for residues located close to the (6-4) photoproduct in DNA, indicating the binding to this region. Intermolecular NOEs between DNA and distamycin A have been identified for some of these residues, suggesting the formation of the relatively tight complex. On the basis of these NOEs, structure determination of the complex is now in progress.

Binding Sites↗

PubMed2005

DNA polymerase iota-dependent translesion replication of uracil containing cyclobutane pyrimidine dimers.

Analysis of the spectrum of UV-induced mutations generated in synchronized wild-type S-phase cells reveals that only approximately 25% of mutations occur at thymine (T), whilst 75% are targeted to cytosine (C). The mutational spectra changes dramatically in XP-V cells, devoid of poleta, where approximately 45% of mutations occur at Ts and approximately 55% at Cs. At the present time, it is unclear whether the C-->T mutations actually represent true misincorporations opposite C, or perhaps occur as the result of the correct incorporation of adenine (A) opposite a C in a UV-photoproduct that had undergone deamination to uracil (U). In order to assess the role that human poliota might play, if any, in the replicative bypass of such UV-photoproducts, we have analyzed the efficiency and fidelity of pol iota-dependent bypass of a T-U cyclobutane pyrimidine dimer (CPD) in vitro. Interestingly, pol iota-dependent bypass of a T-U CPD occurs more efficiently than that of a corresponding T-T CPD. Guanine (G) was misincorporated opposite the 3'U of the T-U CPD only two-fold less frequently than the correct Watson-Crick base, A. While pol iota generally extended the G:3'U-CPD mispairs less efficiently than the correctly paired primer, pol iota-dependent extension was equal to, or greater than that observed with human pols eta and kappa and S. cerevisiae pol zeta under the same assay conditions. Thus, we hypothesize that the ability of pol iota to bypass T-U CPDs through the frequent misincorporation of G opposite the 3'U of the CPD, may provide a mechanism whereby human cells can decrease the mutagenic potential of these lesions.

Carcinogens↗

PubMed2005

Roles of the polymerase and BRCT domains of Rev1 protein in translesion DNA synthesis in yeast in vivo.

Rev1p in yeast is essential for the translesion of abasic sites and 6-4 photoproducts. It plays a role as a translesion polymerase, but also supports translesion catalyzed by other polymerases. The protein has two domains, BRCT and Y-family polymerase. A point mutation in the BRCT domain is known to abolish the second function. In the present research, we have studied the effects of deletion of the BRCT domain and a point mutation at the two amino acids in the putative polymerase active center. We have introduced an abasic site, its tetrahydrofuran analog, and a 6-4 thymine-thymine photoproduct using the oligonucleotide transformation assay. Translesion efficiencies were estimated from the transforming activities of the oligonucleotides with a lesion, and the mutation spectra were analyzed by DNA sequencing of the transformants. Results showed that the lack of the BRCT domain reduced translesion efficiencies, but that substantial translesion synthesis took place. The mutation spectra of the lesions were not greatly affected. Therefore, the BRCT domain may be important, but dispensable for translesion synthesis. In contrast, the polymerase mutation, rev1AA, has only small effects on the translesion efficiencies, but the mutation spectra were greatly affected; the incorporation of dCMP opposite the lesions was specifically lost. This clearly shows that the polymerase domain is responsible for the dCMP incorporation. The effect of Poleta was also analyzed. From all the results DNA polymerases other than these two translesion polymerases, too, seem to initiate the translesion synthesis.

Amino Acid Sequence↗

PubMed2005

DDB1-DDB2 (xeroderma pigmentosum group E) protein complex recognizes a cyclobutane pyrimidine dimer, mismatches, apurinic/apyrimidinic sites, and compound lesions in DNA.

The DDB protein complex, comprising the subunits DDB1 and DDB2, binds tightly to UV light-irradiated DNA. Mutations in DDB2 are responsible for xeroderma pigmentosum group E, a disorder with defects in nucleotide excision repair of DNA. Both subunits are also components of a complex involved in ubiquitin-mediated proteolysis. Cellular defects in DDB2 disable repair of the major UV radiation photoproduct in DNA, a cyclobutane pyrimidine dimer, but no significant direct binding of DDB to this photoproduct in DNA has ever been demonstrated. Thus, it has been uncertain how DDB could play a specific role in DNA repair of such damage. We investigated DDB function using highly purified proteins. Co-purified DDB1-DDB2 or DDB reconstituted with individual DDB1 and DDB2 subunits binds to damaged DNA as a ternary complex. We found that DDB can indeed recognize a cyclobutane pyrimidine dimer in DNA with an affinity (K(app)a) 6-fold higher than that of nondamaged DNA. The DDB1-DDB2 complex also bound with high specificity to a UV radiation-induced (6-4) photoproduct and to an apurinic site in DNA. Unexpectedly, DDB also bound avidly to DNA containing a 2- or 3-bp mismatch (and does not bind well to DNA containing larger mismatches). These data indicate that DDB does not detect lesions per se. It instead recognizes other structural features of damaged DNA, acting as a sensor that probes DNA for a subset of conformational changes. Lesions recognized may include those arising when translesion polymerases such as POLH incorporate bases across from DNA lesions caused by UV radiation.

Amino Acid Sequence↗

PubMed2005

Binding of MutS mismatch repair protein to DNA containing UV photoproducts, "mismatched" opposite Watson--Crick and novel nucleotides, in different DNA sequence contexts.

Mismatch-repair (MMR) systems suppress mutation via correction of DNA replication errors (base-mispairs) and responses to mutagenic DNA lesions. Selective binding of mismatched or damaged DNA by MutS-homolog proteins-bacterial MutS, eukaryotic MSH2.MSH6 (MutSalpha) and MSH2.MSH3-initiates mismatch-correction pathways and responses to lesions, and may cumulatively increase discrimination at downstream steps. MutS-homolog binding selectivity and the well-known but poorly understood effects of DNA-sequence contexts on recognition may thus be primary determinants of MMR specificity and efficiency. MMR processes that modulate UV mutagenesis might begin with selective binding by MutS homologs of "mismatched" T[CPD]T/AG and T[6--4]T/AG photoproducts, reported previously for hMutSalpha and described here for E. coli MutS protein. If MMR suppresses UV mutagenesis by acting directly on pre-mutagenic products of replicative bypass, mismatched photoproducts should be recognized in most DNA-sequence contexts. In three of four contexts tested here (three substantially different), T[CPD]T/AG was bound only slightly better by MutS than was T[CPD]T/AA or homoduplex DNA; only one of two contexts tested promoted selective binding of T[6--4]T/AG. Although the T:G pairs in T[CPD]T/AG and T/G both adopt wobble conformations, MutS bound T/G well in all contexts (K(1/2) 2.1--2.9 nM). Thus, MutS appears to select the two mismatches by different mechanisms. NMR analyses elsewhere suggest that in the (highly distorted) T[6--4]T/AG a forked H-bond between O2 of the 3' thymine and the ring 1-imino and exocyclic 2-amino guanine protons stabilizes a novel planar structure not possible in T[6--4]T/AA. Replacement of G by purines lacking one (inosine, 2-aminopurine) or both (nebularine) protons markedly reduced or eliminated selective MutS binding, as predicted. Previous studies and the work here, taken together, suggest that in only about half of DNA sequence contexts could MutS (and presumably MutSalpha) selectively bind mismatched UV photoproducts and directly suppress UV mutagenesis.

Adenosine Triphosphatases↗

PubMed2005

DNA polymerases eta and kappa are responsible for error-free translesion DNA synthesis activity over a cis-syn thymine dimer in Xenopus laevis oocyte extracts.

In translesion synthesis (TLS), specialized DNA polymerases (pols) facilitate progression of replication forks stalled by DNA damage. Although multiple TLS pols have been identified in eukaryotes, little is known about endogenous TLS pols and their relative contributions to TLS in vivo because of their low cellular abundance. Taking advantage of Xenopus laevis oocyte cells, with their extraordinary size and abundant enzymes involved in DNA metabolism, we have identified and characterized endogenous TLS pols for DNA damage induced by ultraviolet (UV) irradiation. We designed a TLS assay which monitors primer elongation on a synthetic oligomer template over a single UV-induced lesion, either a cys-syn cyclobutane pyrimidine dimer (CPD) or a pyrimidine (6-4) pyrimidone photoproduct. Four distinct TLS activities (TLS1-TLS4) were identified in X. laevis oocyte extracts, using three template/primer (T/P) DNA substrates having various sites at which primer extension is initiated relative to the lesion. TLS1 and TLS2 activities appear to be sequence-dependent. TLS3 and TLS4 extended the primers over the CPD in an error-free manner irrespective of sequence context. Base insertion opposite the CPD of the T/P substrate in which the 3'-end of the primer is placed one base upstream of the lesion was observed only with TLS3. TLS3 and TLS4 showed primer extension with similar efficiencies on the T/P substrate whose 3'-primer terminal dinucleotide (AA) was complementary to the CPD lesion. Investigations with antibodies and recombinant pols revealed that TLS3 and TLS4 were most likely attributable to pol eta and pol kappa, respectively. These results indicate that error-free insertion in CPD bypass is due mainly to pol eta (TLS3) in the extracts, and suggest that pol kappa (TLS4) may assist pol eta (TLS3) in error-free extension during CPD bypass.

Animals↗

PubMed2005

Calculation of fluence to dose conversion coefficients in partial exposure.

Organ doses and effective doses of electrons and photons for the partial exposure using the MIRD-5 phantom and EGS-4 Code have been calculated. Irradiation geometries are AP and PA exposure. Calculations are carried out using 5 isocentered beams with different sizes to investigate the beam sizes effect on the effective dose. The results show that the effective doses depend not only on the energy of incident particle but also on the exposure position and the beam size on the body.

Electrons↗