Tracing the Fate of Site-Specifically Introduced DNA
Damage in the Human Genome
Masamitsu HonmaNational Institute of Health Sciences,
Tokyo, Japan
Studies of Environmental Mutagens
DNA Damage
Mutation
Classical Approach (Retrospective)
Prospective Analysis
Mutagens MutationsDNA Damage
New Strategy in Mutagenesis Research
Adverse Outcome Pathway (AOP)
Prospective Analysis
Introducing a DNA Damage by Gene Targeting and Tracing its Fate in Genome
Target Mutagenesis Oxidative DNA
Damage (8-oxoG)
DNA Double Strand Break
(DSB)
Fate?
(I) (II)
Targeting Mutagenesis Project Started from 2000
• Yasui et al, Tracing the fates of site-specifically introduced DNA adducts in the human genome. DNA Repair 15, 11-20 (2014)
• Yatagai et al., Preliminary results of space experient: Implication for the effects of space radiation and microgravity on survival and mutation induction in human cells. Ad. In Space Res. 49, 479-486 (2012)
• Takashima et al., Dependence of DNA double strand break repair pathways on cell cycle phase in human lymphoblastoid cells. Environ Mol Mutagen. 815-822 (2009)
• Honma et al., Non-homologous end-joining for repairing I-SceI-induced DNA double strand breaks in human cells. DNA Repair, 6, 781-188 (2007)
• Honma et al., Deletion, rearrangement, and gene conversion; the genetic consequences of chromosomal double-strand breaks in human cells. Environ. Mol. Mutagen., 42, 288-298 (2003)
DNA Double Strand Break (DSB) Repair in Mammalian Chromosomes
HR(Allelic)
NHEJ
HR(SCE)
Error-Prone(deletion, etc)
Error-Free (LOH)
Error-Free
G1
S/G2
DNA Replication
IR
exon 5 exon 6 exon 7exon 4 I-Sce I
5‘-AATACCCTGTTAT CCCTA-3’3’-TAATGGGAC AATAGGGAT-5’
I-Sce I site pCBASce vector
Human TK6 Cells
Target Mutagenesis at I-SceI Restriction Enzyme Site
TK gene
TK gene
γ-H2AX Foci at DSBs
I-SceI γ-Ray (2Gy)
I-SceI 2Gy-Ionizing Radiation
Mut
ant f
requ
ency
(X10
-6)
10-0
10-1
10-2
10-3
10-4
10-5
10-6
10-7
TK-Mutant Frequency in TSCE5 and TSCE105 by I-Sce I
Spont γRay(2Gy)
I-SceI X1TSCE5
I-SceI X 2TSCE105
Analysis of Deletion Mutants By PCR
11 12 13 15(kb)1410
I-Sce I
175F 199R
5618 bp
Mar
ker
TSC
E5
Deletion mutants from TSCE5
7000
5000
3000
20001500
1000
(bp)
(Analyzed by Agilent 2100 Bioanalyzer)
TK gene
5618 bp
Clone DNA sequence of TSCE5 mutants around I-SceI site Size(bp)
ori. TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTATCCCTA CTCTCGAGGATCTGGCAG
1659 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTAT-CCTA CTCTCGAGGATCTGGCAG -11841 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTAT-CCTA CTCTCGAGGATCTGGCAG -11893 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTAT-CCTA CTCTCGAGGATCTGGCAG -11875 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT--TATCCCTA CTCTCGAGGATCTGGCAG -22099 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGT---CCCTA CTCTCGAGGATCTGGCAG -32399 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTAT----A CTCTCGAGGATCTGGCAG -41573 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTTAT----A CTCTCGAGGATCTGGCAG -42182 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTA--------TCCCTA CTCTCGAGGATCTGGCAG -82238 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTA--------TCCCTA CTCTCGAGGATCTGGCAG -81678 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -91878 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -91907 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -92003 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -92083 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -92183 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCT---------A CTCTCGAGGATCTGGCAG -92070 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATTACCCTGTT------- --CTCGAGGATCTGGCAG -91657 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC AT-----------CCCTA CTCTCGAGGATCTGGCAG -112078 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC AT---------------- -----------CTGGCAG -281627 TCCGGGCCAAATGGCCGGAGTTGTCAGATCC ATT--------------- ------------TGGCAG -28
No.
12345678910111213141516171819
Analysis of Deletion Mutants by DNA Sequencing
Mutant Clones
Non-homologous End-Joining; NHEJ
Small deletion; <60bp
Large deletion; >60bp
Homologous Recombination; HR
%
93
79
14
7
Summary of Mutational Spectra of I-Sce I Inducing a DSB in TSCE5 Cells
*NHEJ is the major pathway for repairing endonuclease-induced DSBs in mammalian cells.
TSCER122(TK-/- )
6 7exon 4TK-
TK-
exon 5 6 7
pvINTG targeting vector
I-Sce I
Inter-allelic Recombination and Gene-Target Integration Induced by a DSB
Gene Targeting by HR
Inter-allelic Recombination
I-SceII-SceI+
pvINTG
TK re
vert
antf
requ
ency
pvINTG Spont.
10-2
10-8
10-4
10-6
TK revertant frequencies by Inter-allelic Recombination and Gene-Target Integration
Gene Targeting by HR
Inter-allelic Recombination
Tracing DNA adducts in targeted mutagenesis (TATAM)
Yasui M et al., DNA Repair 15, 11-20 (2014)
TK geneTK gene
Genome
TK gene
I-SceI site
I-SceI
pvINT8OxG
TK- TK-
DNA adduct
TK+DNA adductDSB
TSCER122 cell (TK -/-) TSCER122 cell (TK +/-)
IntegratedDNA adduct
A single I-SceI-induced DSB
Construction of targeting vectors containing a single DNA adduct
H H
Cytosine 5-bromocytosine(5-BrC)
H
Guanine
HXanthine
(Xan)
8-bromoguanine(8-BrG)
H
NO
NH
N
N
H
NH2
BrH
8-oxoguanine (8-oxoG)
O
Tracing DNA adducts in targeted mutagenesis (TATAM)
TK-TK-
I-SceI
Ⅵ ⅦⅣ Ⅴ
TSCER122 cell
Ⅵ ⅦⅤ
8-oxoG in BssSI site
TK+
Gene targeting by HR with pvINT8OxG vector
HAT selection and cloning
PCRSequencing
PCR products (821 bps)
MseIRTargeting vector containing a single 8-oxoG (pvINT8OxG)
Determination of mutation frequency and
spectrum of 8-oxoG
Mutation in BssSI site
TK-
Induction of a DSB by pCBASce vector
I-SceI+
pvINTG
I-SceI+
pvINT8oxG
TK re
vert
antf
requ
ency
pvINTG Spont.
10-2
10-8
10-4
10-6
TK revertant frequencies by Gene-Targeting with Intact and 8-oxoG vectors.
No difference of gene targeting efficiency between intact and 8-oxoG vectors.
HH
8-oxoguanine (8-oxoG)
Guanine
O
Mutation frequency and spectrum associated with a single 8-oxoG
X = G T C A Del. Ins. Others ND
Intact (G) 1 206 0 0 0 0 0 3 02 561 551 0 0 0 0 1 9 0
Total 770 (100%) 757 (98.3%) 0 0 0 0 1 (0.1%) 1 (0.1%) 12 08-oxoG 1 207 176 13 2 1 4 2 5 4
2 596 516 34 8 5 13 4 13 3Total 803 (100%) 692 (86%) 47 (5.9%) 10 (1.2%) 6 (0.7%) 17 (2.1%) 6 (0.7%) 86 (10.7%) 18 7
Point mutationNo. of targeted mutants (G or 8-oxoG)
Targetingvector Experiment
Total of pointmutation
No. of revertantsAnalyzed
8-oxoG 1 199 171 5 3 1 7 2 7 32 308 276 8 5 4 8 1 5 1
Total 507(100%) 447 (88%) 13 (2.6%) 8 (1.6%) 5 (1%) 15 (2.9%) 3 (0.6%) 44 (8.7%) 12 4
TSECR122
TSECR122MYH
209
H H
Xanthine(Xan)
Guanine
Mutation frequency and spectrum associated with a single Xan, 8-BrG, and 5-BrC
2 561 551 0 0 0 0 1 9 0Total 770 (100%) 757 (98.3%) 0 0 0 0 1 (0.1%) 1 (0.1%) 12 0
1 287 217 58 1 0 0 0 6 52 260 197 54 3 1 0 0 2 3
Total 547 (100%) 414 (75.7%) 112 (20.5%)4 (0.7%) 1 (0.2%) 0 0 117 (21.4%) 8 8
H H
Cytosine 5-bromocytosine(5-BrC)
1 411 00403 1 2 0 5 02 474 00461 0 1 0 2 0
Total 885 (100%) 00864 (97.6%) 1(0.1%) 3(0.3%) 0 4 (0.4%) 17 0
X = G T C A Del. Ins. Others ND
Intact (G)
Xan
Point mutationNo. of targeted mutants (G, Xan or 5BrC)
Targetingvector Experiment
Total of pointmutation
No. of revertantsAnalyzed
8-BrG
TSECR1221 209 206 0 0 0 0 0 3 0
1 377 37500 0 0 0 1 12 328 32302 0 0 0 1 2
Total 705 (100%) 698 (99%)02 (0.3%) 0 0 0 2 (0.3%) 2 3
5-BrC
8-bromoguanine(8-BrG)
H
NO
NH
N
N
H
NH2
Br
Only single 8-oxoG adduct in the human genome produces a gene mutation at high probability, and causes G to T transversion predominantly.
The oxidative DNA damage does not have threshold to induce gene mutation, and DNA repair mechanism (MYH) does not contribute to establish the threshold of 8-oxoG inducing genotoxicity.
Mutagenicity of DNA adducts; Xan > 8-oxoG > 8-BrG = 5-BrC
Summary of Mutational Spectra of A Single DNA Adduct Induced by TATAM System
Summary of Mutator Phenotype of RecQ2 Knockout TK6 cells by ZFN
CancerTumor
suppressor gene
1st Hit
Loss of Heterozygosity(LOH)
HR
NHEJ
LOH by HR and NHEJ highly occurs in BLM. It is a nature of genomic instability of BLM patient associating with cancer-predisposition.
Acknowledgement
National Institute of Health Sciences
Manabu YasuiAkira SasaNagisa KamoshitaAkiko UkaiSatoshi TadokoroMayumi SakurabaTomoko KoizumiHiroko SakamotoWengsheng WangMakoto HayashiTetsuya SuzukiYuki Kanemaru
Institute of Physical and Chemical Research (RIKEN)
Masako IzumiFumio Yatagai
Toshiya Arakawa
Health Sciences University of Hokkaido